Method for producing butanol using extractive fermentation with electrolyte addition

ABSTRACT

A method for producing butanol through microbial fermentation, in which the butanol product is removed during the fermentation by extraction into a water-immiscible organic extractant in the presence of at least one electrolyte at a concentration at least sufficient to increase the butanol partition coefficient relative to that in the presence of the salt concentration of the basal fermentation medium, is provided. The electrolyte may comprise a salt which dissociates in the fermentation medium, or in the aqueous phase of a biphasic fermentation medium, to form free ions. Also provided is a method and composition for recovering butanol from a fermentation medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/952,480, filed Nov. 23, 2010 which claims the benefit of priority tothe U.S. Provisional Patent Application Ser. No. 61/263,519, filed onNov. 23, 2009, the entirety of which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the field of biofuels. Morespecifically, the invention relates to a method for producing butanolthrough microbial fermentation, in which at least one electrolyte ispresent in the fermentation medium at a concentration at leastsufficient to increase the butanol partition coefficient relative tothat in the presence of the salt concentration of the basal fermentationmedium, and the butanol product is removed by extraction into awater-immiscible organic extractant.

BACKGROUND

Butanol is an important industrial chemical with a variety ofapplications, such as use as a fuel additive, as a blend component todiesel fuel, as a feedstock chemical in the plastics industry, and as afoodgrade extractant in the food and flavor industry. Each year 10 to 12billion pounds of butanol are produced by petrochemical means. As theneed for butanol increases, interest in producing this chemical fromrenewable resources such as corn, sugar cane, or cellulosic feeds byfermentation is expanding.

In a fermentative process to produce butanol, in situ product removaladvantageously reduces butanol inhibition of the microorganism andimproves fermentation rates by controlling butanol concentrations in thefermentation broth. Technologies for in situ product removal includestripping, adsorption, pervaporation, membrane solvent extraction, andliquid-liquid extraction. In liquid-liquid extraction, an extractant iscontacted with the fermentation broth to partition the butanol betweenthe fermentation broth and the extractant phase. The butanol and theextractant are recovered by a separation process, for example bydistillation.

J. J. Malinowski and A. J. Daugulis, AlChE Journal (1994), 40(9),1459-1465, disclose experimental studies to assess the effect of saltaddition on the extraction of 1-butanol, ethanol, and acetone fromdilute aqueous solutions using cyclopentanol, n-valeraldehyde, tert-amylalcohol, and Adol 85NF (comprised largely of oleyl alcohol) asextractants. The authors note in their conclusions that in spite of theadvantages that salt addition offers to the extraction of ethanol,1-butanol, and acetone from dilute aqueous solutions typically found infermentation processes, the practical implementation of such a processconfiguration is presently limited. As an in situ recovery strategy(extractive fermentation) the relatively high salts concentrations whichmay be required could have severely deleterious effects on cells arisingfrom osmotic shock.

Published Patent Application US 2009/0171129 A1 discloses methods forrecovery of C3-C6 alcohols from dilute aqueous solutions, such asfermentation broths. The method includes increasing the activity of theC3-C6 alcohol in a portion of the aqueous solution to at least that ofsaturation of the C3-C6 alcohol in the portion. According to anembodiment of the invention, increasing the activity of the C3-C6alcohol may comprise adding a hydrophilic solute to the aqueoussolution. Sufficient hydrophilic solute is added to enable the formationof a second liquid phase, either solely by addition of the hydrophilicsolute or in combination with other process steps. The added hydrophilicsolute may be a salt, an amino acid, a water-soluble solvent, a sugar orcombinations of those.

U.S. patent application Ser. No. 12/478,389 filed on Jun. 4, 2009,discloses methods for producing and recovering butanol from afermentation broth, the methods comprising the step of contacting thefermentation broth with a water-immiscible organic extractant selectedfrom the group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fattyacids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, andmixtures thereof, to form a two-phase mixture comprising an aqueousphase and a butanol-containing organic phase.

U.S. Provisional Patent Application Nos. 61/168,640; 61/168,642; and61/168,645; filed concurrently on Apr. 13, 2009; and 61/231,697;61/231,698; and 61/231,699; filed concurrently on Aug. 6, 2009, disclosemethods for producing and recovering butanol from a fermentation medium,the methods comprising the step of contacting the fermentation mediumwith a water-immiscible organic extractant comprising a first solventand a second solvent, the first solvent being selected from the groupconsisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, estersof C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, and mixturesthereof, and the second solvent being selected from the group consistingof C₇ to C₁₁ alcohols, C₇ to C₁₁carboxylic acids, esters of C₇ to C₁₁carboxylic acids, C₇ to C₁₁ aldehydes, and mixtures thereof, to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase.

Improved methods for producing and recovering butanol from afermentation medium are continually sought. A process for in situproduct removal of butanol in which electrolyte addition to afermentation medium provides improved butanol extraction efficiency andacceptable biocompatibility with the microorganism is desired.

SUMMARY OF THE INVENTION

The present invention provides a method for recovering butanol from afermentation medium comprising butanol, water, at least one electrolyte,and a genetically modified microorganism that produces butanol from atleast one fermentable carbon source. The electrolyte is present in thefermentation medium at a concentration at least sufficient to increasethe butanol partition coefficient relative to that in the presence ofthe salt concentration of the basal fermentation medium. The presentinvention also provides methods for the production of butanol using sucha microorganism and an added electrolyte. The methods include contactingthe fermentation medium with i) a first water-immiscible organicextractant and optionally ii) a second water-immiscible organicextractant, separating the butanol-containing organic phase from theorganic phase, and recovering the butanol from the butanol-containingorganic phase. In one embodiment of the invention, a method forrecovering butanol from a fermentation medium is provided, the methodcomprising:

a) providing a fermentation medium comprising butanol, water, at leastone electrolyte at a concentration at least sufficient to increase thebutanol partition coefficient relative to that in the presence of thesalt concentration of the basal fermentation medium, and a geneticallymodified microorganism that produces butanol from at least onefermentable carbon source;

b) contacting the fermentation medium with i) a first water-immiscibleorganic extractant selected from the group consisting of C₁₂ to C₂₂fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fattyacids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides and mixturesthereof, and optionally ii) a second water-immiscible organic extractantselected from the group consisting of C₇ to C₂₂ fatty alcohols, C₇ toC₂₂ fatty acids, esters of C₇ to C₂₂ fatty acids, C₇ to C₂₂ fattyaldehydes, C₇ to C₂₂ fatty amides and mixtures thereof to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase;

c) optionally, separating the butanol-containing organic phase from theaqueous phase; and

d) recovering the butanol from the butanol-containing organic phase toproduce recovered butanol.

In some embodiments, a portion of the butanol is concurrently removedfrom the fermentation medium by a process comprising the steps of: a)stripping butanol from the fermentation medium with a gas to form abutanol-containing gas phase; and b) recovering butanol from thebutanol-containing gas phase.

According to the methods of the invention, the electrolyte may be addedto the fermentation medium, to the first extractant, to the optionalsecond extractant, or to combinations thereof. In some embodiments, theelectrolyte comprises a salt having a cation selected from the groupconsisting of lithium, sodium, potassium, rubidium, cesium, magnesium,calcium, strontium, barium, ammonium, phosphonium, and combinationsthereof. In some embodiments, the electrolyte comprises a salt having ananion selected from the group consisting of sulfate, carbonate, acetate,citrate, lactate, phosphate, fluoride, chloride, bromide, iodide, andcombinations thereof. In some embodiments, the electrolyte is selectedfrom the group consisting of sodium sulfate, sodium chloride, andcombinations thereof.

According to the methods of the invention, in some embodiments thegenetically modified microorganism is selected from the group consistingof bacteria, cyanobacteria, filamentous fungi, and yeasts. In someembodiments, the bacteria are selected from the group consisting ofZymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus,Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella,Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium. Insome embodiments the yeast is selected from the group consisting ofPichia, Candida, Hansenula, Kluyveromyces, Issatchenkia, andSaccharomyces.

According to the methods of the invention, the first extractant may beselected from the group consisting of oleyl alcohol, behenyl alcohol,cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleicacid, lauric acid, myristic acid, stearic acid, methyl myristate, methyloleate, lauric aldehyde, 1-dodecanol, and a combination of these. Insome embodiments, the first extractant comprises oleyl alcohol. In someembodiments, the second extractant may be selected from the groupconsisting of 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal,and a combination of these.

In some embodiments, the butanol is 1-butanol. In some embodiments, thebutanol is 2-butanol. In some embodiments, the butanol is isobutanol. Insome embodiments, the fermentation medium further comprises ethanol, andthe butanol-containing organic phase contains ethanol.

In one embodiment of the invention, a method for the production ofbutanol is provided, the method comprising:

a) providing a genetically modified microorganism that produces butanolfrom at least one fermentable carbon source;

b) growing the microorganism in a biphasic fermentation mediumcomprising an aqueous phase and i) a first water-immiscible organicextractant selected from the group consisting of C₁₂ to C₂₂ fattyalcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides and mixtures thereof,and optionally ii) a second water-immiscible organic extractant selectedfrom the group consisting of C₇ to C₂₂ alcohols, C₇ to C₂₂carboxylicacids, esters of C₇ to C₂₂ carboxylic acids, C₇ to C₂₂ aldehydes, C₇ toC₂₂ fatty amides, and mixtures thereof, wherein the biphasicfermentation medium further comprises at least one electrolyte at aconcentration at least sufficient to increase the butanol partitioncoefficient relative to that in the presence of the salt concentrationof the basal fermentation medium, for a time sufficient to allowextraction of the butanol into the organic extractant to form abutanol-containing organic phase;

c) optionally, separating the butanol-containing organic phase from theaqueous phase; and

d) recovering the butanol from the butanol-containing organic phase toproduce recovered butanol.

In one embodiment of the invention, a method for the production ofbutanol is provided, the method comprising:

a) providing a genetically modified microorganism that produces butanolfrom at least one fermentable carbon source;

b) growing the microorganism in a fermentation medium wherein themicroorganism produces the butanol into the fermentation medium toproduce a butanol-containing fermentation medium;

c) adding at least one electrolyte to the fermentation medium to providethe electrolyte at a concentration at least sufficient to increase thebutanol partition coefficient relative to that in the presence of thesalt concentration of the basal fermentation medium;

d) contacting at least a portion of the butanol-containing fermentationmedium with i) a first water-immiscible organic extractant selected fromthe group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fattyacids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂to C₂₂ fatty amides and mixtures thereof, and optionally ii) a secondwater-immiscible organic extractant selected from the group consistingof C₇ to C₂₂ alcohols, C₇ to C₂₂ carboxylic acids, esters of C₇ to C₂₂carboxylic acids, C₇ to C₂₂ aldehydes, C₇ to C₂₂ fatty amides, andmixtures thereof, to form a two-phase mixture comprising an aqueousphase and a butanol-containing organic phase;

e) optionally, separating the butanol-containing organic phase from theaqueous phase;

f) recovering the butanol from the butanol-containing organic phase; and

g) optionally, returning at least a portion of the aqueous phase to thefermentation medium.

In some embodiments, the genetically modified microorganism comprises amodification which inactivates a competing pathway for carbon flow. Insome embodiments the genetically modified microorganism does not produceacetone.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

FIG. 1 schematically illustrates one embodiment of the methods of theinvention, in which the first extractant and the second extractant arecombined in a vessel prior to contacting with the fermentation medium ina fermentation vessel.

FIG. 2 schematically illustrates one embodiment of the methods of theinvention, in which the first extractant and the second extractant areadded separately to a fermentation vessel in which the fermentationmedium is contacted with the extractants.

FIG. 3 schematically illustrates one embodiment of the methods of theinvention, in which the first extractant and the second extractant areadded separately to different fermentation vessels.

FIG. 4 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first extractant and the second extractant arecombined in a vessel prior to contacting the fermentation medium withthe extractants in a different vessel.

FIG. 5 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first extractant and the second extractant are addedseparately to a vessel in which the fermentation medium is contactedwith the extractants.

FIG. 6 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs downstream of thefermentor and the first extractant and the second extractant are addedseparately to different vessels for contacting with the fermentationmedium.

FIG. 7 schematically illustrates one embodiment of the methods of theinvention, in which extraction of the product occurs in at least onebatch fermentor via co-current flow of a water-immiscible organicextractant at or near the bottom of a fermentation mash to fill thefermentor with extractant which flows out of the fermentor at a point ator near the top of the fermentor.

The following sequences conform with 37 C.F.R. 1.821 1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a bis), and Section 208 and Annex C of theAdministrative Instructions).

TABLE 1a SEQ ID Numbers of Coding Sequences and Proteins SEQ ID NO: SEQID NO: Description Nucleic acid Amino acid Klebsiella pneumonias budB(acetolactate 1 2 synthase) E. coli ilvC (acetohydroxy acid 3 4reductoisomerase) E. coli ilvD (acetohydroxy acid 5 6 dehydratase)Lactococcus lactis kivD (branched-chain α- 7 (codon 8 keto aciddecarboxylase) optimized) Achromobacter xylosoxidans sadB 9 10 (butanoldehydrogenase) Bacillus subtilis alsS (acetolactate 11 12 synthase) S.cerevisiae ILV5 (acetohydroxy acid 13 14 reductoisomerase; “KARI”)Mutant KARI (encoded by Pf5.ilvC-Z4B8) 15 16 Streptococcus mutans ilvD(acetohydroxy 17 18 acid dehydratase) Bacillus subtilis kivD(branched-chain keto 19 (codon 20 acid decarboxylase) optimized) Horseliver alcohol dehydrogenase 56 (codon 57 (HADH) optimized) E. coli pflB(pyruvate formate lyase) 71 70 E. coli frdB (subunit of fumaratereductase 73 72 enzyme complex) E. coli ldhA (lactate dehydrogenase) 7776 E. coli adhE (alcohol dehydrogenase) 75 74 E. coli frdA (subunit offumarate reductase 91 90 enzyme complex) E. coli frdC (subunit offumarate reductase 93 92 enzyme complex) E. coli frdD (subunit offumarate reductase 95 94 enzyme complex)

TABLE 1b SEQ ID Numbers of Sequences used in construction, Primers andVectors Description SEQ ID NO: pRS425::GPM-sadB 63 GPM-sadB-ADHt segment21 pUC19-URA3r 22 114117-11A 23 114117-11B 24 114117-11C 25 114117-11D26 114117-13A 27 114117-13B 28 112590-34F 29 112590-34G 30 112590-34H 31112590-49E 32 ilvD-FBA1t segment 33 114117-27A 34 114117-27B 35114117-27C 36 114117-27D 37 114117-36D 38 135 39 112590-30F 40 URA3r2template 41 114117-45A 42 114117-45B 43 PDC5::KanMXF 44 PDC5::KanMXR 45PDC5kofor 46 N175 47 pLH475-Z4B8 plasmid 48 CUP1 promoter 49 CYC1terminator CYC1-2 50 ILV5 promoter 51 ILV5 terminator 52 FBA1 promoter53 CYC1 terminator 54 pLH468 plasmid 55 Vector pNY8 58 GPD1 promoter 59GPD1 promoter fragment 60 OT1068 61 OT1067 62 GPM1 promoter 64 ADH1terminator 65 OT1074 66 OT1075 67 pRS423 FBA ilvD(Strep) 68 FBAterminator 69 pflB CkUp 78 pflB CkDn 79 frdB CkUp 80 frdB CkDn 81 ldhACkUp 82 ldhA CkDn 83 adhE CkUp 84 adhE CkDn 85 N473 86 N469 87 N695A 88N695B 89

DETAILED DESCRIPTION

The present invention provides methods for recovering butanol from amicrobial fermentation medium comprising at least one electrolyte byextraction into a water-immiscible organic extractant to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase. The electrolyte is present in the fermentation medium ata concentration at least sufficient to increase the butanol partitioncoefficient relative to that in the presence of the salt concentrationof the basal fermentation medium. The butanol-containing organic phaseis separated from the aqueous phase and the butanol may be recovered.Methods for producing butanol are also provided.

DEFINITIONS

The following definitions are used in this disclosure.

The term “electrolyte” refers to a solute that ionizes or dissociates inan aqueous solution and may function as an ionic conductor.

The term “butanol” refers to 1-butanol, 2-butanol, and/or isobutanol,individually or as mixtures thereof.

The term “water-immiscible” refers to a chemical component, such as anextractant or solvent, which is incapable of mixing with an aqueoussolution, such as a fermentation broth, in such a manner as to form oneliquid phase.

The term “extractant” as used herein refers to one or more organicsolvents which are used to extract butanol from a fermentation broth.

The term “biphasic fermentation medium” refers to a two-phase growthmedium comprising a fermentation medium (i.e., an aqueous phase) and asuitable amount of a water-immiscible organic extractant.

The term “organic phase”, as used herein, refers to the non-aqueousphase of a biphasic mixture obtained by contacting a fermentation brothwith a water-immiscible organic extractant.

The term “aqueous phase”, as used herein, refers to the phase of abiphasic mixture, obtained by contacting an aqueous fermentation mediumwith a water-immiscible organic extractant, which comprises water.

The term “In Situ Product Removal” as used herein means the selectiveremoval of a specific fermentation product from a biological processsuch as fermentation to control the product concentration in thebiological process.

The term “fermentation broth” as used herein means the mixture of water,sugars, dissolved solids, suspended solids, microorganisms producingbutanol, product butanol and all other constituents of the material heldin the fermentation vessel in which product butanol is being made by thereaction of sugars to butanol, water and carbon dioxide (CO₂) by themicroorganisms present. The fermentation broth may comprise one or morefermentable carbon sources such as the sugars described herein. Thefermentation broth is the aqueous phase in biphasic fermentativeextraction. From time to time, as used herein the term “fermentationmedium” may be used synonymously with “fermentation broth”.

The term “fermentation vessel” as used herein means the vessel in whichthe fermentation reaction by which product butanol is made from sugarsis carried out. The term “fermentor” may be used synonymously hereinwith “fermentation vessel”.

The term “fermentable carbon source” refers to a carbon source capableof being metabolized by the microorganisms disclosed herein. Suitablefermentable carbon sources include, but are not limited to,monosaccharides, such as glucose or fructose; disaccharides, such aslactose or sucrose; oligosaccharides; polysaccharides, such as starch orcellulose; one-carbon substrates; and a combination of these, which maybe found in the fermentation medium. Sources of fermentable carboninclude renewable carbon, that is non-petroleum-based carbon, includingcarbon from agricultural feedstocks, algae, cellulose, hemicellulose,lignocellulose, or any combination thereof.

The term “fatty acid” as used herein refers to a carboxylic acid havinga long, aliphatic chain of C₇ to C₂₂ carbon atoms, which is eithersaturated or unsaturated.

The term “fatty alcohol” as used herein refers to an alcohol having along, aliphatic chain of C₇ to C₂₂ carbon atoms, which is eithersaturated or unsaturated.

The term “fatty aldehyde” as used herein refers to an aldehyde having along, aliphatic chain of C₇ to C₂₂ carbon atoms, which is eithersaturated or unsaturated.

The term “fatty amide” as used herein refers to an amide having a long,aliphatic chain of C₁₂ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “partition coefficient”, abbreviated herein as K_(p), means theratio of the concentration of a compound in the two phases of a mixtureof two immiscible solvents at equilibrium. A partition coefficient is ameasure of the differential solubility of a compound between twoimmiscible solvents. As used herein, the term “partition coefficient forbutanol” refers to the ratio of concentrations of butanol between theorganic phase comprising the extractant and the aqueous phase comprisingthe fermentation medium. Partition coefficient, as used herein, issynonymous with the term distribution coefficient.

The term “separation” as used herein is synonymous with “recovery” andrefers to removing a chemical compound from an initial mixture to obtainthe compound in greater purity or at a higher concentration than thepurity or concentration of the compound in the initial mixture.

The term “butanol biosynthetic pathway” as used herein refers to anenzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.

The term “1-butanol biosynthetic pathway” as used herein refers to anenzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).

The term “2-butanol biosynthetic pathway” as used herein refers to anenzyme pathway to produce 2-butanol from pyruvate.

The term “isobutanol biosynthetic pathway” as used herein refers to anenzyme pathway to produce isobutanol from pyruvate.

The term “effective titer” as used herein, refers to the total amount ofbutanol produced by fermentation per liter of fermentation medium. Thetotal amount of butanol includes: (i) the amount of butanol in thefermentation medium; (ii) the amount of butanol recovered from theorganic extractant; and (iii) the amount of butanol recovered from thegas phase, if gas stripping is used.

The term “effective rate” as used herein, refers to the total amount ofbutanol produced by fermentation per liter of fermentation medium perhour of fermentation.

The term “effective yield” as used herein, refers to the amount ofbutanol produced per unit of fermentable carbon substrate consumed bythe biocatalyst

The term “aerobic conditions” as used herein means growth conditions inthe presence of oxygen.

The term “microaerobic conditions” as used herein means growthconditions with low levels of oxygen (i.e., below normal atmosphericoxygen levels).

The term “anaerobic conditions” as used herein means growth conditionsin the absence of oxygen.

The term “minimal media” as used herein refers to growth media thatcontain the minimum nutrients possible for growth, generally without thepresence of amino acids. A minimal medium typically contains afermentable carbon source and various salts, which may vary amongmicroorganisms and growing conditions; these salts generally provideessential elements such as magnesium, nitrogen, phosphorus, and sulfurto allow the microorganism to synthesize proteins and nucleic acids.

The term “defined media” as used herein refers to growth media that haveknown quantities of all ingredients present, e.g., a defined carbonsource and nitrogen source, and trace elements and vitamins required bythe microorganism.

The term “biocompatibility” as used herein refers to the measure of theability of a microorganism to utilize glucose in the presence of anextractant. A biocompatible extractant permits the microorganism toutilize glucose. A non-biocompatible (that is, a biotoxic) extractantdoes not permit the microorganism to utilize glucose, for example at arate greater than about 25% of the rate when the extractant is notpresent.

The term, “° C.” means degrees Celsius. The term “OD” means opticaldensity. The term “OD₆₀₀” refers to the optical density at a wavelengthof 600 nm. The term ATCC refers to the American Type Culture Collection,Manassas, Va. The term “sec” means second(s). The term “min” meansminute(s). The term “h” means hour(s). The term “mL” meansmilliliter(s). The term “L” means liter. The term “g” means grams. Theterm “mmol” means millimole(s). The term “M” means molar. The term “μL”means microliter. The term “μg” means microgram. The term “μg/mL” meansmicrogram per liter. The term “mL/min” means milliliters per minute. Theterm “g/L” means grams per liter. The term “g/L/h” means grams per literper hour. The term “mmol/min/mg” means millimole per minute permilligram. The term “temp” means temperature. The term “rpm” meansrevolutions per minute. The term “HPLC” means high pressure gaschromatography. The term “GC” means gas chromatography.

All publications, patents, patent applications, and other referencesmentioned herein are expressly incorporated by reference in theirentireties for all purposes. Further, when an amount, concentration, orother value or parameter is given as either a range, preferred range, ora list of upper preferable values and lower preferable values, this isto be understood as specifically disclosing all ranges formed from anypair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

Genetically Modified Microorganisms

Microbial hosts for butanol production may be selected from bacteria,cyanobacteria, filamentous fungi and yeasts. The microbial host usedshould be tolerant to the butanol product produced, so that the yield isnot limited by toxicity of the product to the host. The selection of amicrobial host for butanol production is described in detail below.

Microbes that are metabolically active at high titer levels of butanolare not well known in the art. Although butanol-tolerant mutants havebeen isolated from solventogenic Clostridia, little information isavailable concerning the butanol tolerance of other potentially usefulbacterial strains. Most of the studies on the comparison of alcoholtolerance in bacteria suggest that butanol is more toxic than ethanol(de Cavalho et al., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz etal., FEMS Microbiol. Lett. 220:223-227 (2003)). Tomas et al. (J.Bacteriol. 186:2006-2018 (2004)) report that the yield of 1-butanolduring fermentation in Clostridium acetobutylicum may be limited bybutanol toxicity. The primary effect of 1-butanol on Clostridiumacetobutylicum is disruption of membrane functions (Hermann et al.,Appl. Environ. Microbiol. 50:1238-1243 (1985)).

The microbial hosts selected for the production of butanol should betolerant to butanol and should be able to convert carbohydrates tobutanol using an introduced biosynthetic pathway, such as the pathwaydescribed below. The criteria for selection of suitable microbial hostsinclude the following: intrinsic tolerance to butanol, high rate ofcarbohydrate utilization, availability of genetic tools for genemanipulation, and the ability to generate stable chromosomalalterations.

Suitable host strains with a tolerance for butanol may be identified byscreening based on the intrinsic tolerance of the strain. The intrinsictolerance of microbes to butanol may be measured by determining theconcentration of butanol that is responsible for 50% inhibition of thegrowth rate (IC50) when grown in a minimal medium. The IC50 values maybe determined using methods known in the art. For example, the microbesof interest may be grown in the presence of various amounts of butanoland the growth rate monitored by measuring the optical density at 600nanometers. The doubling time may be calculated from the logarithmicpart of the growth curve and used as a measure of the growth rate. Theconcentration of butanol that produces 50% inhibition of growth may bedetermined from a graph of the percent inhibition of growth versus thebutanol concentration. Preferably, the host strain should have an IC50for butanol of greater than about 0.5%. More suitable is a host strainwith an IC50 for butanol that is greater than about 1.5%. Particularlysuitable is a host strain with an IC50 for butanol that is greater thanabout 2.5%.

The microbial host for butanol production should also utilize glucoseand/or other carbohydrates at a high rate. Most microbes are capable ofutilizing carbohydrates. However, certain environmental microbes cannotefficiently use carbohydrates, and therefore would not be suitablehosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. Modes of gene transfertechnology that may be used include by electroporation, conjugation,transduction or natural transformation. A broad range of hostconjugative plasmids and drug resistance markers are available. Thecloning vectors used with an organism are tailored to the host organismbased on the nature of antibiotic resistance markers that can functionin that host.

The microbial host also may be manipulated in order to inactivatecompeting pathways for carbon flow by inactivating various genes. Thisrequires the availability of either transposons or chromosomalintegration vectors to direct inactivation. Additionally, productionhosts that are amenable to chemical mutagenesis may undergo improvementsin intrinsic butanol tolerance through chemical mutagenesis and mutantscreening.

As an example of inactivation of competing pathways for carbon flow,pyruvate decarboxylase may be reduced or eliminated (see, for example,US Patent Application Publication No. 20090305363). In embodiments,butanol is the major product of the microorganism. In embodiments, themicroorganism does not produce acetone.

Based on the criteria described above, suitable microbial hosts for theproduction of butanol include, but are not limited to, members of thegenera, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas,Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula, Kluyveromyces, Issatchenkia,and Saccharomyces. Preferred hosts include: Escherichia coli,Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans,Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum,Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis,Pediococcus pentosaceus, Pediococcus acidilactici, Bacillus subtilis andSaccharomyces cerevisiae.

Microorganisms mentioned above may be genetically modified to convertfermentable carbon sources into butanol, specifically 1-butanol,2-butanol, or isobutanol, using methods known in the art. Suitablemicroorganisms include Escherichia, Lactobacillus, and Saccharomyces.Suitable microorganisms include E. coli, L. plantarum and S. cerevisiae.Additionally, the microorganism may be a butanol-tolerant strain of oneof the microorganisms listed above that is isolated using the methoddescribed by Bramucci et al. (U.S. patent application Ser. No.11/761,497; and WO 2007/146377). An example of one such strain isLactobacillus plantarum strain PN0512 (ATCC: PTA-7727, biologicaldeposit made Jul. 12, 2006 for U.S. patent application Ser. No.11/761,497).

Suitable biosynthetic pathways for production of butanol are known inthe art, and certain suitable pathways are described herein. In someembodiments, the butanol biosynthetic pathway comprises at least onegene that is heterologous to the host cell. In some embodiments, thebutanol biosynthetic pathway comprises more than one gene that isheterologous to the host cell. In some embodiments, the butanolbiosynthetic pathway comprises heterologous genes encoding polypeptidescorresponding to every step of a biosynthetic pathway.

Likewise, certain suitable proteins having the ability to catalyzeindicated substrate to product conversions are described herein andother suitable proteins are provided in the art. For example, US PatentApplication Publication Nos. US20080261230, US20090163376, andUS20100197519 describe acetohydroxy acid isomeroreductases as does U.S.application Ser. No. 12/893,077, filed on Sep. 29, 2010; US PatentApplication Publication No. 20100081154 describes dihydroxyaciddehydratases; alcohol dehydrogenases are described in US PatentApplication Publication No. US20090269823 and U.S. Provisional PatentApplication No. 61/290,636.

Microorganisms can be genetically modified to contain a 1-butanolbiosynthetic pathway to produce 1-butanol. Suitable modificationsinclude those described by Donaldson et al. in WO 2007/041269. Forexample, the microorganism may be genetically modified to express a1-butanol biosynthetic pathway comprising the following enzyme-catalyzedsubstrate to product conversions:

a) acetyl-CoA to acetoacetyl-CoA;

b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA;

c) 3-hydroxybutyryl-CoA to crotonyl-CoA;

d) crotonyl-CoA to butyryl-CoA;

e) butyryl-CoA to butyraldehyde; and

f) butyraldehyde to a-butanol.

The microorganisms may also be genetically modified to express a2-butanol biosynthetic pathway to produce 2-butanol. Suitablemodifications include those described by Donaldson et al. in U.S. PatentApplication Publication Nos. 2007/0259410 and 2007/0292927, and PCTApplication Publication Nos. WO 2007/130518 and WO 2007/130521. Forexample, in one embodiment the microorganism may be genetically modifiedto express a 2-butanol biosynthetic pathway comprising the followingenzyme-catalyzed substrate to product conversions:

a) pyruvate to alpha-acetolactate;

b) alpha-acetolactate to acetoin;

c) acetoin to 2,3-butanediol;

d) 2,3-butanediol to 2-butanone; and

e) 2-butanone to 2-butanol.

The microorganisms may also be genetically modified to express anisobutanol biosynthetic pathway to produce isobutanol. Suitablemodifications include those described by Donaldson et al. in U.S. PatentApplication Publication No. 2007/0092957 and WO 2007/050671. Forexample, the microorganism may be genetically modified to contain anisobutanol biosynthetic pathway comprising the followingenzyme-catalyzed substrate to product conversions:

a) pyruvate to acetolactate;

b) acetolactate to 2,3-dihydroxyisovalerate;

c) 2,3-dihydroxyisovalerate to α-ketoisovalerate;

d) α-ketoisovalerate to isobutyraldehyde; and

e) isobutyraldehyde to isobutanol.

The Escherichia coli strain may comprise: (a) an isobutanol biosyntheticpathway encoded by the following genes: budB (SEQ ID NO:1) fromKlebsiella pneumoniae encoding acetolactate synthase (given as SEQ IDNO:2), ilvC (given as SEQ ID NO:3) from E. coli encoding acetohydroxyacid reductoisomerase (given as SEQ ID NO:4), ilvD (given as SEQ IDNO:5) from E. coli encoding acetohydroxy acid dehydratase (given as SEQID NO:6), kivD (given as SEQ ID NO:7) from Lactococcus lactis encodingthe branched-chain keto acid decarboxylase (given as SEQ ID NO:8), andsadB (given as SEQ ID NO:9) from Achromobacter xylosoxidans encoding abutanol dehydrogenase (given as SEQ ID NO:10). The enzymes encoded bythe genes of the isobutanol biosynthetic pathway catalyze the substrateto product conversions for converting pyruvate to isobutanol, asdescribed above. Specifically, acetolactate synthase catalyzes theconversion of pyruvate to acetolactate, acetohydroxy acidreductoisomerase catalyzes the conversion of acetolactate to2,3-dihydroxyisovalerate, acetohydroxy acid dehydratase catalyzes theconversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate,branched-chain keto acid decarboxylase catalyzes the conversion ofα-ketoisovalerate to isobutyraldehyde, and butanol dehydrogenasecatalyzes the conversion of isobutyraldehyde to isobutanol. Thisrecombinant Escherichia coli strain can be constructed using methodsknown in the art (see copending U.S. patent application Ser. Nos.12/478,389 and 12/477,946) and/or described herein below. It iscontemplated that suitable strains may be constructed comprising asequence having at least about 70-75% identity, at least about 75-80%,at least about 80-85% identity, or at least about 85-90% identity toprotein sequences described herein.

The Escherichia coli strain may comprise deletions of the followinggenes to eliminate competing pathways that limit isobutanol production,pflB, given as SEQ ID No: 71, (encoding for pyruvate formate lyase)IdhA, given as SEQ IS NO: 73, (encoding for lactate dehydrogenase),adhE, given as SEQ IS NO: 77, (encoding for alcohol dehydrogenase), andat least one gene comprising the frdABCD operon (encoding for fumaratereductase), specifically, frdA, given as SEQ ID NO: 90, frdB, given asSEQ ID NO: 75, frdC, given as SEQ ID NO: 92, and frdD, given as SEQ IDNO: 94.

The Saccharomyces cerevisiae strain may comprise: an isobutanolbiosynthetic pathway encoded by the following genes: alsS coding regionfrom Bacillus subtilis (SEQ ID NO:11) encoding acetolactate synthase(SEQ ID NO:12), ILV5 from S. cerevisiae (SEQ ID NO:13) encodingacetohydroxy acid reductoisomerase (KARI; SEQ ID NO:14) and/or a mutantKARI such as encoded by Pf5.IlvC-Z4B8 (SEQ ID NO: 15; protein SEQ ID NO:16), ilvD from Streptococcus mutans (SEQ ID NO: 17) encodingacetohydroxy acid dehydratase (SEQ ID NO: 18), kivD from Bacillussubtilis (codon optimized sequence given as SEQ ID NO: 19) encoding thebranched-chain keto acid decarboxylase (SEQ ID NO:20), and sadB fromAchromobacter xylosoxidans (SEQ ID NO:9) encoding a butanoldehydrogenase (SEQ ID NO:10). The enzymes encoded by the genes of theisobutanol biosynthetic pathway catalyze the substrate to productconversions for converting pyruvate to isobutanol, as described herein.It is contemplated that suitable strains may be constructed comprising asequence having at least about 70-75% identity, at least about 75-80%,at least about 80-85% identity, or at least about 85-90% identity toprotein sequences described herein.

A yeast strain expressing an isobutanol pathway with acetolactatesynthase (ALS) activity in the cytosol and deletions of the endogenouspyruvate decarboxylase (PDC) genes is described in U.S. patentapplication Ser. No. 12/477,942. This combination of cytosolic ALS andreduced PDC expression has been found to greatly increase flux frompyruvate to acetolactate, which then flows to the pathway for productionof isobutanol. Such a recombinant Saccharomyces cerevisiae strain can beconstructed using methods known in the art and/or described herein.Other suitable yeast strains are known in the art. Additional examplesare provided in U.S. Provisional Application Ser. Nos. 61/379,546,61/380,563, and U.S. application Ser. No. 12/893,089.

Additional modifications suitable for microorganisms used in conjunctionwith the processes provided herein include modifications to reduceglycerol-3-phosphate dehydrogenase activity as described in US PatentApplication Publication No. 20090305363, modifications to a host cellthat provide for increased carbon flux through an Entner-DoudoroffPathway or reducing equivalents balance as described in US PatentApplication Publication No. 20100120105. Yeast strains with increasedactivity of heterologous proteins that require binding of an Fe—Scluster for their activity are described in US Application PublicationNo. 20100081179. Other modifications include modifications in anendogenous polynucleotide encoding a polypeptide having dual-rolehexokinase activity, described in U.S. Provisional Application No.61/290,639, integration of at least one polynucleotide encoding apolypeptide that catalyzes a step in a pyruvate-utilizing biosyntheticpathway described in U.S. Provisional Application No. 61/380,563.

Additionally, host cells comprising at least one deletion, mutation,and/or substitution in an endogenous gene encoding a polypeptideaffecting Fe—S cluster biosynthesis are described in U.S. ProvisionalPatent Application No. 61/305,333, and host cells comprising aheterologous polynucleotide encoding a polypeptide with phosphoketolaseactivity and host cells comprising a heterologous polynucleotideencoding a polypeptide with phosphotransacetylase activity are describedin U.S. Provisional Patent Application No. 61/356,379.

Construction of a Suitable Yeast Strain

NGI-049 is an example of a suitable Saccharomyces cerevisiae strain.NGI-049 is a strain with insertion-inactivation of endogenous PDC1,PDC5, and PDC6 genes, and containing expression vectors pLH475-Z4B8 andpLH468. PDC1, PDC5, and PDC6 genes encode the three major isozymes ofpyruvate decarboxylase. The strain expresses genes encoding enzymes foran isobutanol biosynthetic pathway that are integrated or on plasmids.Construction of the NGI-049 strain is provided herein.

Endogenous pyruvate decarboxylase activity in yeast converts pyruvate toacetaldehyde, which is then converted to ethanol or to acetyl-CoA viaacetate. Therefore, endogenous pyruvate decarboxylase activity is atarget for reduction or elimination of byproduct formation.

Examples of other yeast strains with reduced pyruvate decarboxylaseactivity due to disruption of pyruvate decarboxylase encoding genes havebeen reported such as for Saccharomyces in Flikweert et al. (Yeast(1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol.(1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann,(Mol Gen Genet. (1993) 241:657-666). Saccharomyces strains having nopyruvate decarboxylase activity are available from the ATCC (Accession#200027 and #200028).

Construction of pdc6::GPMp1-sadB Integration Cassette and PDC6 Deletion:

A pdc6::GPM1p-sadB-ADH1t-URA3r integration cassette was made by joiningthe GPM-sadB-ADHt segment (SEQ ID NO:21) from pRS425::GPM-sadB (SEQ IDNO: 63) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:22)contains the URA3 marker from pRS426 (ATCC #77107) flanked by 75 bphomologous repeat sequences to allow homologous recombination in vivoand removal of the URA3 marker. The two DNA segments were joined by SOEPCR (as described by Horton et al. (1989) Gene 77:61-68) using astemplate pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion DNApolymerase (New England Biolabs Inc., Beverly, Mass.; catalog no.F-540S) and primers 114117-11A through 114117-11D (SEQ ID NOs:23, 24, 25and 26), and 114117-13A and 114117-13B (SEQ ID NOs:27 and 28).

The outer primers for the SOE PCR (114117-13A and 114117-13B) contained5′ and 3′ ˜50 bp regions homologous to regions upstream and downstreamof the PDC6 promoter and terminator, respectively. The completedcassette PCR fragment was transformed into BY4700 (ATCC #200866) andtransformants were maintained on synthetic complete media lacking uraciland supplemented with 2% glucose at 30° C. using standard genetictechniques (Methods in Yeast Genetics, 2005, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformantswere screened by PCR using primers 112590-34G and 112590-34H (SEQ IDNOs:30 and 31), and 112590-34F and 112590-49E (SEQ ID NOs: 29 and 32) toverify integration at the PDC6 locus with deletion of the PDC6 codingregion. The URA3r marker was recycled by plating on synthetic completemedia supplemented with 2% glucose and 5-FOA at 30° C. followingstandard protocols. Marker removal was confirmed by patching coloniesfrom the 5-FOA plates onto SD-URA media to verify the absence of growth.The resulting identified strain has the genotype: BY4700pdc6::P_(GPM1)-sadB-ADH1t.

Construction of pdc1::PDC1-ilvD Integration Cassette and PDC1 Deletion:

A pdc1::PDC1p-ilvD-FBA1t-URA3r integration cassette was made by joiningthe ilvD-FBA1t segment (SEQ ID NO:33) from pLH468 to the URA3r gene frompUC19-URA3r by SOE PCR (as described by Horton et al. (1989) Gene77:61-68) using as template pLH468 and pUC19-URA3r plasmid DNAs, withPhusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.;catalog no. F-540S) and primers 114117-27A through 114117-27D (SEQ IDNOs:34, 35, 36 and 37).

The outer primers for the SOE PCR (114117-27A and 114117-27D) contained5′ and 3′ ˜50 bp regions homologous to regions downstream of the PDC1promoter and downstream of the PDC1 coding sequence. The completedcassette PCR fragment was transformed into BY4700pdc6::P_(GPM1)-sadB-ADH1t and transformants were maintained on syntheticcomplete media lacking uracil and supplemented with 2% glucose at 30° C.using standard genetic techniques (Methods in Yeast Genetics, 2005, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202).Transformants were screened by PCR using primers 114117-36D and 135 (SEQID NOs 38 and 39), and primers 112590-49E and 112590-30F (SEQ ID NOs 32and 40) to verify integration at the PDC1 locus with deletion of thePDC1 coding sequence. The URA3r marker was recycled by plating onsynthetic complete media supplemented with 2% glucose and 5-FOA at 30°C. following standard protocols. Marker removal was confirmed bypatching colonies from the 5-FOA plates onto SD-URA media to verify theabsence of growth. The resulting identified strain “NYLA67” has thegenotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t.

HIS3 Deletion

To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette wasPCR-amplified from URA3r2 template DNA (SEQ ID NO; 41). URA3r2 containsthe URA3 marker from pRS426 (ATCC #77107) flanked by 500 bp homologousrepeat sequences to allow homologous recombination in vivo and removalof the URA3 marker. PCR was done using Phusion DNA polymerase andprimers 114117-45A and 114117-45B (SEQ ID NOs: 42 and 43) whichgenerated a ˜2.3 kb PCR product. The HIS3 portion of each primer wasderived from the 5′ region upstream of the HIS3 promoter and 3′ regiondownstream of the coding region such that integration of the URA3r2marker results in replacement of the HIS3 coding region. The PCR productwas transformed into NYLA67 using standard genetic techniques (Methodsin Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., pp. 201-202) and transformants were selected onsynthetic complete media lacking uracil and supplemented with 2% glucoseat 30° C. Transformants were screened to verify correct integration byreplica plating of transformants onto synthetic complete media lackinghistidine and supplemented with 2% glucose at 30° C. The URA3r markerwas recycled by plating on synthetic complete media supplemented with 2%glucose and 5-FOA at 30° C. following standard protocols. Marker removalwas confirmed by patching colonies from the 5-FOA plates onto SD-URAmedia to verify the absence of growth. The resulting identified strain“NYLA73” has the genotype: BY4700 pdc6::GPM1p-sadB-ADH1tpdc1::PDC1p-ilvD-FBA1t Δhis3.

Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134Wchromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase andprimers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs:44 and 45) whichgenerated a ˜2.2 kb PCR product. The PDC5 portion of each primer wasderived from the 5′ region upstream of the PDC5 promoter and 3′ regiondownstream of the coding region such that integration of the kanMX4marker results in replacement of the PDC5 coding region. The PCR productwas transformed into NYLA73 using standard genetic techniques (Methodsin Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., pp. 201-202) and transformants were selected on YPmedia supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C.Transformants were screened by PCR to verify correct integration at thePDC locus with replacement of the PDC5 coding region using primersPDC5kofor and N175 (SEQ ID NOs: 46 and 47). The identified correcttransformants have the genotype: BY4700 pdc6::GPM1p-sadB-ADH1tpdc1::PDC1p-ilvD-FBA1t Δhis3 pdc5::kanMX4.

pLH475-Z4B8 Construction

The pLH475-Z4B8 plasmid (SEQ ID NO:48) was constructed for expression ofALS and KARI in yeast. pLH475-Z4B8 is a pHR81 vector (ATCC #87541)containing the following chimeric genes: 1) the CUP1 promoter (SEQ IDNO: 49), acetolactate synthase coding region from Bacillus subtilis(AlsS; SEQ ID NO: 11; protein SEQ ID NO: 12) and CYC1 terminator(CYC1-2; SEQ ID NO: 50); 2) an ILV5 promoter (SEQ ID NO:51),Pf5.IlvC-Z4B8 coding region (SEQ ID NO: 15; protein SEQ ID NO: 16) andILV5 terminator (SEQ ID NO:52); and 3) the FBA1 promoter (SEQ ID NO:53), S. cerevisiae KARI coding region (ILV5; SEQ ID NO: 13; protein SEQID NO:14) and CYC1 terminator (SEQ ID NO: 54).

The Pf5.IlvC-Z4B8 coding region is a sequence encoding KARI derived fromPseudomonas fluorescens but containing mutations, that was described inUS Patent Application Publication No. US20090163376. The Pf5.IlvC-Z4B8encoded KARI (SEQ ID NO:16) has the following amino acid changes ascompared to the natural Pseudomonas fluorescens KARI:

C33L: cysteine at position 33 changed to leucine,

R47Y: arginine at position 47 changed to tyrosine,

S50A: serine at position 50 changed to alanine,

T52D: threonine at position 52 changed to asparagine,

V53A: valine at position 53 changed to alanine,

L61F: leucine at position 61 changed to phenylalanine,

T80I: threonine at position 80 changed to isoleucine,

A156V: alanine at position 156 changed to threonine, and

G170A: glycine at position 170 changed to alanine.

The Pf5.IlvC-Z4B8 coding region was synthesized by DNA 2.0 (Palo Alto,Calif.; SEQ ID NO:15) based on codons that were optimized for expressionin Saccharomyces cerevisiae.

Expression Vector pLH468

The pLH468 plasmid (SEQ ID NO: 55) was constructed for expression ofDHAD, KivD and HADH in yeast.

Coding regions for B. subtilis ketoisovalerate decarboxylase (KivD) andHorse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0based on codons that were optimized for expression in Saccharomycescerevisiae (SEQ ID NO:19 and 56, respectively) and provided in plasmidspKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs 20and 57, respectively. Individual expression vectors for KivD and HADHwere constructed. To assemble pLH467 (pRS426::P_(GPD1)-kivDy-GPD1t),vector pNY8 (SEQ ID NO:58; also named pRS426.GPD-ald-GPDt, described inUS Patent App. Pub. US20080182308, Example 17) was digested with AscIand SfiI enzymes, thus excising the GPD1 promoter (SEQ ID NO: 59) andthe ald coding region. A GPD1 promoter fragment (GPD1-2; SEQ ID NO: 60)from pNY8 was PCR amplified to add an AscI site at the 5′ end, and anSpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067(SEQ ID NOs: 61 and 62). The AscI/SfiI digested pNY8 vector fragment wasligated with the GPD1 promoter PCR product digested with AscI and SpeI,and the SpeI-SfiI fragment containing the codon optimized kivD codingregion isolated from the vector pKivD-DNA2.0. The triple ligationgenerated vector pLH467 (PRS426::P_(GPD1)-kivDy-GPD1t). pLH467 wasverified by restriction mapping and sequencing.

pLH435 (pRS425::P_(GPM1)-Hadhy-ADH1t) was derived from vectorpRS425::GPM-sadB (SEQ ID NO:63) which is described in U.S. patentapplication Ser. No. 12/477,942, Example 3. pRS425::GPM-sadB is thepRS425 vector (ATCC #77106) with a chimeric gene containing the GPM1promoter (SEQ ID NO:64), coding region from a butanol dehydrogenase ofAchromobacter xylosoxidans (sadB; SEQ ID NO: 9; protein SEQ ID NO:10:disclosed in US Patent App. Publication No. US20090269823), and ADH1terminator (SEQ ID NO:65). pRS425::GPMp-sadB contains BbvI and PacIsites at the 5′ and 3′ ends of the sadB coding region, respectively. ANheI site was added at the 5′ end of the sadB coding region bysite-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NO:66and 67) to generate vector pRS425-GPMp-sadB-NheI, which was verified bysequencing. pRS425::P_(GPM1)-sadB-NheI was digested with NheI and PacIto drop out the sadB coding region, and ligated with the NheI-PacIfragment containing the codon optimized HADH coding region from vectorpHadhy-DNA2.0 to create pLH435.

To combine KivD and HADH expression cassettes in a single vector, yeastvector pRS411 (ATCC #87474) was digested with SacI and NotI, and ligatedwith the SacI-SalI fragment from pLH467 that contains theP_(GPD1)-kivDy-GPD1t cassette together with the SalI-NotI fragment frompLH435 that contains the P_(GPM1)-Hadhy-ADH1t cassette in a tripleligation reaction. This yielded the vectorpRS411::P_(GPD1)-kivDy-P_(GPM1)-Hadhy (pLH441), which was verified byrestriction mapping.

In order to generate a co-expression vector for all three genes in thelower isobutanol pathway: ilvD, kivDy and Hadhy, we used pRS423 FBAilvD(Strep) (SEQ ID NO:68), which is described in U.S. patentapplication Ser. No. 12/569,636 as the source of the IlvD gene. Thisshuttle vector contains an F1 origin of replication (nt 1423 to 1879)for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) forreplication in yeast. The vector has an FBA promoter (nt 2111 to 3108;SEQ ID NO: 53;) and FBA terminator (nt 4861 to 5860; SEQ ID NO: 69). Inaddition, it carries the H is marker (nt 504 to 1163) for selection inyeast and ampicillin resistance marker (nt 7092 to 7949) for selectionin E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO: 17;protein SEQ ID NO: 18) from Streptococcus mutans UA159 (ATCC #700610) isbetween the FBA promoter and FBA terminator forming a chimeric gene forexpression. In addition there is a lumio tag fused to the ilvD codingregion (nt 4829-4849).

The first step was to linearize pRS423 FBA ilvD(Strep) (also calledpRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio) with SacI and SacII(with SacII site blunt ended using T4 DNA polymerase), to give a vectorwith total length of 9,482 bp. The second step was to isolate thekivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI siteblunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment.This fragment was ligated with the 9,482 bp vector fragment frompRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio. This generated vectorpLH468(pRS423::P_(FBA1)-ilvD(Strep)Lumio-FBA1t-P_(GPD1)-kivDy-GPD1t-P_(GPM1)-hadhy-ADH1t),which was confirmed by restriction mapping and sequencing.

Plasmid vectors pLH468 and pLH475-Z4B8 were simultaneously transformedinto strain BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3pdc5::kanMX4 using standard genetic techniques (Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) and the resulting strain was maintained on synthetic completemedia lacking histidine and uracil, and supplemented with 1% ethanol at30° C. The resulting strain was named NGI-049.

Organic Extractants

The extractant is a water-immiscible organic solvent or solvent mixturehaving characteristics which render it useful for the extraction ofbutanol from a fermentation broth. A suitable organic extractant shouldmeet the criteria for an ideal solvent for a commercial two-phaseextractive fermentation for the production or recovery of butanol.Specifically, the extractant should (i) be biocompatible with themicroorganisms, for example Escherichia coli, Lactobacillus plantarum,and Saccharomyces cerevisiae, (ii) be substantially immiscible with thefermentation medium, (iii) have a high partition coefficient (K_(P)) forthe extraction of butanol, (iv) have a low partition coefficient for theextraction of nutrients, (v) have a low tendency to form emulsions withthe fermentation medium, and (vi) be low cost and nonhazardous. Inaddition, for improved process operability and economics, the extractantshould (vii) have low viscosity (μ), (viii) have a low density (ρ)relative to the aqueous fermentation medium, and (ix) have a boilingpoint suitable for downstream separation of the extractant and thebutanol.

In one embodiment, the extractant may be biocompatible with themicroorganism, that is, nontoxic to the microorganism or toxic only tosuch an extent that the microorganism is impaired to an acceptablelevel, so that the microorganism continues to produce the butanolproduct into the fermentation medium. The extent of biocompatibility ofan extractant can be determined by the glucose utilization rate of themicroorganism in the presence of the extractant and the butanol product,as measured under defined fermentation conditions. See, for example, theExamples in U.S. Provisional Patent Application Nos. 61/168,640;61/168,642; and 61/168,645. While a biocompatible extractant permits themicroorganism to utilize glucose, a non-biocompatible extractant doesnot permit the microorganism to utilize glucose at a rate greater than,for example, about 25% of the rate when the extractant is not present.As the presence of the fermentation product butanol can affect thesensitivity of the microorganism to the extractant, the fermentationproduct should be present during biocompatibility testing of theextractant. The presence of additional fermentation products, forexample ethanol, may similarly affect the biocompatibility of theextractant. Use of a biocompatible extractant is desired for processesin which continued production of butanol is desired after contacting thefermentation broth comprising the microorganism with an organicextractant.

In one embodiment, the extractant may be selected from the groupconsisting of C₇ to C₂₂ fatty alcohols, C₇ to C₂₂ fatty acids, esters ofC₇ to C₂₂ fatty acids, C₇ to C₂₂ fatty aldehydes, C₇ to C₂₂ fattyamides, and mixtures thereof. Examples of suitable extractants includean extractant comprising at least one solvent selected from the groupconsisting of oleyl alcohol, behenyl alcohol, cetyl alcohol, laurylalcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid,myristic acid, stearic acid, methyl myristate, methyl oleate, lauricaldehyde, 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal,2-butyloctanol, 2-butyl-octanoic acid and mixtures thereof. Inembodiments, the extractant comprises oleyl alcohol. In embodiments, theextractant comprises a branched chain saturated alcohol, for example,2-butyloctanol, commercially available as ISOFAL® 12 (Sasol, Houston,Tex.) or Jarcol I-12 (Jarchem Industries, Inc., Newark, N.J.). Inembodiments, the extractant comprises a branched chain carboxylic acid,for example, 2-butyl-octanoic acid, 2-hexyl-decanoic acid, or2-decyl-tetradecanoic acid, commercially available as ISOCARB® 12,ISOCARB® 16, and ISOCARB® 24, respectively (Sasol, Houston, Tex.).

In one embodiment, a first water-immiscible organic extractant may beselected from the group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ toC₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fattyaldehydes, C₁₂ to C₂₂ fatty amides, and mixtures thereof. Suitable firstextractants may be further selected from the group consisting of oleylalcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol also referred toas 1-dodecanol, myristyl alcohol, stearyl alcohol, oleic acid, lauricacid, myristic acid, stearic acid, methyl myristate, methyl oleate,lauric aldehyde, and mixtures thereof. In one embodiment, the extractantmay comprise oleyl alcohol.

In one embodiment, an optional second water-immiscible organicextractant may be selected from the group consisting of C₇ to C₂₂ fattyalcohols, C₇ to C₂₂ fatty carboxylic acids, esters of C₇ to C₂₂ fattycarboxylic acids, C₇ to C₂₂ fatty aldehydes, C₇ to C₂₂ fatty amides, andmixtures thereof. Suitable second extractants may be further selectedfrom the group consisting of 1-nonanol, 1-decanol, 1-undecanol,2-undecanol, 1-nonanal, and mixtures thereof. In one embodiment, thesecond extractant comprises 1-decanol.

In one embodiment, the first extractant comprises oleyl alcohol and thesecond extractant comprises 1-decanol.

When a first and a second extractant are used, the relative amounts ofeach can vary within a suitable range. For example, the first extractantmay be used in an amount which is about 30 percent to about 90 percent,or about 40 percent to about 80 percent, or about 45 percent to about 75percent, or about 50 percent to about 70 percent of the combined volumeof the first and the second extractants. The optimal range reflectsmaximization of the extractant characteristics, for example balancing arelatively high partition coefficient for butanol with an acceptablelevel of biocompatibility. For a two-phase extractive fermentation forthe production or recovery of butanol, the temperature, contacting time,butanol concentration in the fermentation medium, relative amounts ofextractant and fermentation medium, specific first and secondextractants used, relative amounts of the first and second extractants,presence of other organic solutes, the presence and concentration ofelectrolytes, and the amount and type of microorganism are related; thusthese variables may be adjusted as necessary within appropriate limitsto optimize the extraction process as described herein.

Suitable organic extractants may be available commercially from varioussources, such as Sigma-Aldrich (St. Louis, Mo.), in various grades, manyof which may be suitable for use in extractive fermentation to produceor recover butanol. Technical grades of a solvent can contain a mixtureof compounds, including the desired component and higher and lowermolecular weight components. For example, one commercially availabletechnical grade oleyl alcohol contains about 65% oleyl alcohol and amixture of higher and lower fatty alcohols.

Electrolyte

According to the present method, the fermentation medium contains atleast one electrolyte at a concentration at least sufficient to increasethe butanol partition coefficient relative to that in the presence ofthe salt concentration of the basal fermentation medium. The electrolytemay comprise one or more of the salts contained in the basalfermentation medium, in which case the electrolyte is present at aconcentration above that of the concentration of the total saltscontained in the basal fermentation medium. The electrolyte may compriseone or more salts which are not present in the basal fermentationmedium. The basal fermentation medium may contain, for example,phosphate, magnesium, and/or ammonium salts and is generally tailored toa specific microorganism. Suggested compositions of basal fermentationmedia may be found in Difco™ & BBL™ manual (Becton Dickinson andCompany, Sparks, Md. 21152, USA). Generally, the salts provided by traceelements may be ignored in the calculation of the total saltconcentration of the basal fermentation medium due to their extremelylow concentrations.

The electrolyte may comprise a salt which dissociates in thefermentation medium, or in the aqueous phase of a biphasic fermentationmedium, to form free ions. For example, the electrolyte may comprise asalt having a cation selected from the group consisting of lithium,sodium, potassium, rubidium, cesium, magnesium, calcium, strontium,barium, ammonium, phosphonium, and combinations thereof. For example,the electrolyte may comprise a salt having an anion selected from thegroup consisting of sulfate, carbonate, acetate, citrate, lactate,phosphate, fluoride, chloride, bromide, iodide, and combinationsthereof. The electrolyte may be selected from the group consisting ofsodium sulfate, sodium chloride, and combinations thereof.

The electrolyte may be available commercially from various sources, suchas Sigma-Aldrich (St. Louis, Mo.), in various grades, many of which maybe suitable for use in extractive fermentation to produce or recoverbutanol by the methods disclosed herein. The electrolyte may berecovered by methods know in the art from a fermentation medium or froman aqueous phase formed by contacting the fermentation medium with anextractant or other physical or chemical methods such as precipitation,crystallization, and/or evaporation. The recovered electrolyte may beused in a subsequent fermentation.

The amount of electrolyte needed to achieve a concentration in thefermentation medium at least sufficient to increase the butanolpartition coefficient relative to that in the presence of the saltconcentration of the basal fermentation medium can be determined asdisclosed, for example, by the procedures of the Examples herein below.The range of electrolyte concentrations which have a positive effect onthe partition coefficient is determined, for example by experimentation.The range of electrolyte concentrations which demonstrate acceptablebiocompatibility with the microorganism of interest is also determined.The range of suitable electrolyte concentrations are then selected fromthe overlap of these two ranges, such that the amount of electrolyterequired to have a positive effect on the butanol partition coefficientis balanced with the concentration range that provides an acceptablelevel of biocompatibility with the microorganism. Economicconsiderations may also be a factor in selecting the amount of osmolyteto use.

In one embodiment, the electrolyte may be present in the fermentationmedium at a concentration which is biocompatible with the microorganism,that is, nontoxic to the microorganism or toxic only to such an extentthat the microorganism is impaired to an acceptable level, so that themicroorganism continues to produce the butanol product into thefermentation medium in the presence of the electrolyte. The extent ofbiocompatibility of an electrolyte can be determined by the growth rateof the microorganism in the presence of varying concentrations of theelectrolyte, as described in Example 2 herein below. While abiocompatible electrolyte concentration permits the microorganism toutilize glucose (or other carbon source) or grow, a non-biocompatibleelectrolyte concentration does not permit the microorganism to utilizeglucose (or other carbon source) or grow at a rate greater than, forexample, about 25% of the growth rate when the excess amount ofelectrolyte is not present. The presence of fermentation products, forexample butanol, may also affect the concentration ranges of theelectrolyte which have biocompatibility with the microorganism. Use ofan electrolyte within concentration ranges having biocompatibility isdesired for processes in which continued production of butanol isnecessary after contacting the fermentation medium comprising themicroorganism with the electrolyte. In processes in which continuedproduction of butanol after contacting the fermentation mediumcomprising the microorganism with the electrolyte is not required, anelectrolyte may be used at concentration ranges which have little, ifany, biocompatibility with the microorganism.

To achieve a concentration in the fermentation medium of electrolytewhich is at least sufficient to increase the butanol partitioncoefficient relative to that in the presence of the salt concentrationof the basal fermentation medium, the electrolyte may be added to thefermentation medium or to the aqueous phase of a biphasic fermentationmedium during the growth phase of the microorganism, during the butanolproduction phase, when the butanol concentration is inhibitory, or tocombinations thereof. The electrolyte may be added to the firstextractant, to the second extractant, or to combinations thereof. Theelectrolyte may be added as a solid, as a slurry, or as an aqueoussolution. Optionally, the electrolyte may be added to both thefermentation medium and the extractant(s). The electrolyte may be addedin a continuous, semi-continuous, or batch manner. The electrolyte maybe added to the entire stream to which it is introduced, for example tothe entire fermentation medium in a fermentor, or to a partial streamtaken from one or more vessels, for example to a partial stream takenfrom a fermentor.

In embodiments, the total concentration of electrolyte in thefermentation medium is greater than about 0.05M, 0.1M, 0.2M, 0.3M, 0.4M,0.5M, 0.6M, 0.7M, 0.8M, or 1M. In some embodiments, the concentration ofelectrolyte in the fermentation is less than about 1M, and in someembodiments, the concentration of electrolyte in the fermentation isless than 2M.

Fermentation

The microorganism may be cultured in a suitable fermentation medium in asuitable fermentor to produce butanol. Any suitable fermentor may beused including a stirred tank fermentor, an airlift fermentor, a bubblefermentor, or any combination thereof. Materials and methods for themaintenance and growth of microbial cultures are well known to thoseskilled in the art of microbiology or fermentation science (see forexample, Bailey et al., Biochemical Engineering Fundamentals, secondedition, McGraw Hill, New York, 1986). Consideration must be given toappropriate fermentation medium, pH, temperature, and requirements foraerobic, microaerobic, or anaerobic conditions, depending on thespecific requirements of the microorganism, the fermentation, and theprocess. The fermentation medium used is not critical, but it mustsupport growth of the microorganism used and promote the biosyntheticpathway necessary to produce the desired butanol product. A conventionalfermentation medium may be used, including, but not limited to, complexmedia containing organic nitrogen sources such as yeast extract orpeptone and at least one fermentable carbon source; minimal media; anddefined media. Suitable fermentable carbon sources include, but are notlimited to, monosaccharides, such as glucose or fructose; disaccharides,such as lactose or sucrose; oligosaccharides; polysaccharides, such asstarch or cellulose; one carbon substrates; and mixtures thereof. Inaddition to the appropriate carbon source, the fermentation medium maycontain a suitable nitrogen source, such as an ammonium salt, yeastextract or peptone, minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art (Bailey et al., supra).Suitable conditions for the extractive fermentation depend on theparticular microorganism used and may be readily determined by oneskilled in the art using routine experimentation.

Methods for Recovering Butanol Using Extractive Fermentation with AddedElectrolyte

Butanol may be recovered from a fermentation medium containing butanol,water, at least one electrolyte at a concentration at least sufficientto increase the butanol partition coefficient relative to that in thepresence of the salt concentration of the basal fermentation medium,optionally at least one fermentable carbon source, and a microorganismthat has been genetically modified (that is, genetically engineered) toproduce butanol via a biosynthetic pathway from at least one carbonsource. Such genetically modified microorganisms can be selected frombacteria, cyanobacteria, filamentous fungi and yeasts and includeEscherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae,for example. One step in the process is contacting the fermentationmedium with a first water-immiscible organic extractant and optionally asecond water-immiscible organic extractant to form a two-phase mixturecomprising an aqueous phase and a butanol-containing organic phase.“Contacting” means the fermentation medium and the organic extractant orits solvent components are brought into physical contact at any timeduring the fermentation process. The electrolyte may be added to thefermentation medium, to the first extractant, to the optional secondextractant, or to combinations thereof. In one embodiment, thefermentation medium further comprises ethanol, and thebutanol-containing organic phase can contain ethanol.

When a first and a second extractant are used, the contacting may beperformed with the first and second extractants having been previouslycombined. For example, the first and second extractants may be combinedin a vessel such as a mixing tank, and the combined extractants may thenbe added to a vessel containing the fermentation medium. Alternatively,the contacting may be performed with the first and second extractantsbecoming combined during the contacting. For example, the first andsecond extractants may be added separately to a vessel which containsthe fermentation medium. In one embodiment, contacting the fermentationmedium with the organic extractant further comprises contacting thefermentation medium with the first extractant prior to contacting thefermentation medium and the first extractant with the second extractant.In one embodiment, the contacting with the second extractant may occurin the same vessel as the contacting with the first extractant. In oneembodiment, the contacting with the second extractant may occur in adifferent vessel from the contacting with the first extractant. Forexample, the first extractant may be contacted with the fermentationmedium in one vessel, and the contents transferred to another vessel inwhich contacting with the second extractant occurs. In theseembodiments, the electrolyte may be added to the fermentation medium, tothe first extractant, to the optional second extractant, or tocombinations thereof.

The organic extractant may contact the fermentation medium at the startof the fermentation forming a biphasic fermentation medium.Alternatively, the organic extractant may contact the fermentationmedium after the microorganism has achieved a desired amount of growth,which can be determined by measuring the optical density of the culture.In one embodiment, the first extractant may contact the fermentationmedium in one vessel, and the second extractant may contact thefermentation medium and the first extractant in the same vessel. Inanother embodiment, the second extractant may contact the fermentationmedium and the first extractant in a different vessel from that in whichthe first extractant contacts the fermentation medium. In theseembodiments, the electrolyte may be added to the fermentation medium, tothe first extractant, to the optional second extractant, or tocombinations thereof.

Further, the organic extractant may contact the fermentation medium at atime at which the butanol level in the fermentation medium reaches apreselected level, for example, before the butanol concentration reachesa toxic or an inhibitory level. The butanol concentration may bemonitored during the fermentation using methods known in the art, suchas by gas chromatography or high performance liquid chromatography. Theelectrolyte may be added to the fermentation medium before or after thebutanol concentration reaches a toxic or an inhibitory level. Inembodiments, the organic extractant comprises fatty acids. Inembodiments, processes described herein can be used in conjunction withprocesses described in U.S. Provisional Patent Application Nos.61/368,429 and 61/379,546 wherein butanol is esterified with an organicacid such as fatty acid using a catalyst such as a lipase to formbutanol esters.

Fermentation may be run under aerobic conditions for a time sufficientfor the culture to achieve a preselected level of growth, as determinedby optical density measurement. The electrolyte may be added to thefermentation broth before or after the preselected level of growth isachieved. An inducer may then be added to induce the expression of thebutanol biosynthetic pathway in the modified microorganism, andfermentation conditions are switched to microaerobic or anaerobicconditions to stimulate butanol production, as described in detail inExample 6 of U.S. patent application Ser. No. 12/478,389. The extractantmay be added after the switch to microaerobic or anaerobic conditions.The electrolyte may be added before or after the switch to microaerobicor anaerobic conditions. In one embodiment, the first extractant maycontact the fermentation medium prior to the contacting of thefermentation medium and the first extractant with the second extractant.For example, in a batch fermentation process, a suitable period of timemay be allowed to elapse between contacting the fermentation medium withthe first and the second extractants. In a continuous fermentationprocess, contacting the fermentation medium with the first extractantmay occur in one vessel, and contacting of that vessel's contents withthe second extractant may occur in a second vessel. In theseembodiments, the electrolyte may be added to the fermentation medium, tothe first extractant, to the optional second extractant, or tocombinations thereof.

After contacting the fermentation medium with the organic extractant inthe presence of the electrolyte, the butanol product partitions into theorganic extractant, decreasing the concentration in the aqueous phasecontaining the microorganism, thereby limiting the exposure of theproduction microorganism to the inhibitory butanol product. The volumeof the organic extractant to be used depends on a number of factors,including the volume of the fermentation medium, the size of thefermentor, the partition coefficient of the extractant for the butanolproduct, the electrolyte concentration, and the fermentation modechosen, as described below. The volume of the organic extractant may beabout 3% to about 60% of the fermentor working volume. The ratio of theextractant to the fermentation medium is from about 1:20 to about 20:1on a volume:volume basis, for example from about 1:15 to about 15:1, orfrom about 1:12 to about 12:1, or from about 1:10 to about 10:1, or fromabout 1:9 to about 9:1, or from about 1:8 to about 8:1.

The amount of the electrolyte to be added depends on a number offactors, including the effect of the added electrolyte on the growthproperties of the butanol producing microorganism and the effect of theadded electrolyte on the Kp of butanol in a two phase fermentation. Theoptimum amount of electrolyte to be added may also be dependent on thecomposition of the initial basal fermentation medium. Too high aconcentration of an electrolyte, although possibly increasing the Kp ofbutanol and alleviating the toxicity effects of butanol on themicroorganism, can itself be inhibitory to the microorganism. On theother hand, too low a concentration of electrolyte might not increasethe Kp of butanol sufficiently to alleviate the inhibitory effect ofbutanol on the microorganism. Therefore, a balance needs to be foundthrough experimentation to ensure that the net effect of adding excesselectrolyte to the fermentation medium results in an overall increase inthe rate and titer of butanol production. In addition, one couldmodulate the biocompatibility of the salts to the microorganism byaddition of osmoprotectants or osmolytes either exogenously to themedium or by genetically modifying the microorganism to endogenouslyproduce the osmolyte(s). In embodiments, the Kp is increased by about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 100%, about 150%, or about 200% as comparedto the Kp without added electrolyte. In embodiments, the Kp is increasedby at least about 2-fold, at least about 3-fold, at least about 4 fold,at least about 5-fold, or at least about 6-fold. In embodiments, thetotal concentration of electrolyte is selected to increase the Kp by anamount while maintaining the growth rate of the microorganism at a levelthat is at least about 25%, at least about 50%, at least about 80%, orat least about 90% of the growth rate in the absence of addedelectrolyte. In embodiments, the total concentration of electrolyte inthe fermentation medium is sufficient to increase the effective rate ofbutanol production by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, or at leastabout 100% as compared to the rate without added electrolyte. Inembodiments, the total concentration of electrolyte in the fermentationmedium is sufficient to increase the effective yield of butanol by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 100% as comparedto the effective yield without added electrolyte. In embodiments, thetotal concentration of electrolyte in the fermentation medium issufficient to increase the effective titer of butanol by at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or at least about 100% as compared to theeffective titer without added electrolyte.

In embodiments, the amount of added electrolyte is sufficient to resultin an effective titer of at least about 7 g/L, at least about 10 g/L, atleast about 15 g/L, at least about 20 g/L, at least about 25 g/L, atleast about 30 g/L, or at least about 40 g/L. In embodiments, the amountof added electrolyte is sufficient to result in an effective yield of atleast about 0.12, at least about 0.15, at least about 0.2, at leastabout 0.25, or at least about 0.3. In embodiments, the amount of addedelectrolyte is sufficient to result in an effective rate of at leastabout 0.1 g/L/h, at least about 0.15 g/L/h, at least about 0.2 g/L/h, atleast about 0.3 g/L/h, at least about 0.4 g/L/h, at least about 0.6g/L/h, at least about 0.8 g/L/h, at least about 1 g/L/h, or at leastabout 1.2 g/L/h. In some embodiments, the effective rate is about 1.3g/L/h.

The next step is optionally separating the butanol-containing organicphase from the aqueous phase using methods known in the art, includingbut not limited to, siphoning, decantation, centrifugation, using agravity settler, and membrane-assisted phase splitting.

Recovery of the butanol from the butanol-containing organic phase may bedone using methods known in the art, including but not limited to,distillation, adsorption by resins, separation by molecular sieves, andpervaporation. Specifically, distillation may be used to recover thebutanol from the butanol-containing organic phase. The extractant may berecycled to the butanol production and/or recovery process.

The electrolyte may be recovered from the fermentation medium or fromthe aqueous phase of a two phase mixture by methods known in the art.For example, the aqueous phase or fermentation medium may beconcentrated by distillation, stripping, pervaporation, or other methodsto obtain a concentrated aqueous mixture comprising the electrolyte.Optionally, the electrolyte may be returned to a fermentation medium andthus be recycled within the fermentation process. Optionally, theelectrolyte obtained from a fermentation medium may be added to afermentation medium to provide a concentration at least sufficient toincrease the butanol partition coefficient relative to that in thepresence of the salt concentration of the basal fermentation medium.

Gas stripping may be used concurrently with the organic extractant andthe addition of electrolyte to remove the butanol product from thefermentation medium. Gas stripping may be done by passing a gas such asair, nitrogen, or carbon dioxide through the fermentation medium,thereby forming a butanol-containing gas phase. The butanol product maybe recovered from the butanol-containing gas phase using methods knownin the art, such as using a chilled water trap to condense the butanol,or scrubbing the gas phase with a solvent.

Any butanol remaining in the fermentation medium after the fermentationrun is completed may be recovered by continued extraction using fresh orrecycled organic extractant. Alternatively, the butanol can be recoveredfrom the fermentation medium using methods known in the art, such asdistillation, azeotropic distillation, liquid-liquid extraction,adsorption, gas stripping, membrane evaporation, pervaporation, and thelike. In the case where the fermentation medium is not recycled to theprocess, additional electrolyte may be added to further increase thebutanol partition coefficient and improve the efficiency of butanolrecovery.

The two-phase extractive fermentation method may be carried out in acontinuous mode in a stirred tank fermentor. In this mode, the mixtureof the fermentation medium and the butanol-containing organic extractantis removed from the fermentor. The two phases are separated by meansknown in the art including, but not limited to, siphoning, decantation,centrifugation, using a gravity settler, membrane-assisted phasesplitting, and the like, as described above. After separation, thefermentation medium and the electrolyte therein may be recycled to thefermentor or may be replaced with fresh medium, to which additionalelectrolyte is added. Then, the extractant is treated to recover thebutanol product as described above. The extractant may then be recycledback into the fermentor for further extraction of the product.Alternatively, fresh extractant may be continuously added to thefermentor to replace the removed extractant. This continuous mode ofoperation offers several advantages. Because the product is continuallyremoved from the reactor, a smaller volume of organic extractant isrequired enabling a larger volume of the fermentation medium to be used.This results in higher production yields. The volume of the organicextractant may be about 3% to about 50% of the fermentor working volume;3% to about 20% of the fermentor working volume; or 3% to about 10% ofthe fermentor working volume. It is beneficial to use the smallestamount of extractant in the fermentor as possible to maximize the volumeof the aqueous phase, and therefore, the amount of cells in thefermentor. The process may be operated in an entirely continuous mode inwhich the extractant is continuously recycled between the fermentor anda separation apparatus and the fermentation medium is continuouslyremoved from the fermentor and replenished with fresh medium. In thisentirely continuous mode, the butanol product is not allowed to reachthe critical toxic concentration and fresh nutrients are continuouslyprovided so that the fermentation may be carried out for long periods oftime. The apparatus that may be used to carryout these modes oftwo-phase extractive fermentations are well known in the art. Examplesare described, for example, by Kollerup et al. in U.S. Pat. No.4,865,973.

Batchwise fermentation mode may also be used. Batch fermentation, whichis well known in the art, is a closed system in which the composition ofthe fermentation medium is set at the beginning of the fermentation andis not subjected to artificial alterations during the process. In thismode, the desired amount of supplemental electrolyte and a volume oforganic extractant are added to the fermentor and the extractant is notremoved during the process. The organic extractant may be formed in thefermentor by separate addition of the first and the optional secondextractants, or the first and second extractants may be combined to formthe extractant prior to the addition of any extractant to the fermentor.The electrolyte may be added to the fermentation medium, to the firstextractant, to the optional second extractant, or to combinationsthereof. Although this fermentation mode is simpler than the continuousor the entirely continuous modes described above, it requires a largervolume of organic extractant to minimize the concentration of theinhibitory butanol product in the fermentation medium. Consequently, thevolume of the fermentation medium is less and the amount of productproduced is less than that obtained using the continuous mode. Thevolume of the organic extractant in the batchwise mode may be 20% toabout 60% of the fermentor working volume; or 30% to about 60% of thefermentor working volume. It is beneficial to use the smallest volume ofextractant in the fermentor as possible, for the reason described above.

Fed-batch fermentation mode may also be used. Fed-batch fermentation isa variation of the standard batch system, in which the nutrients, forexample glucose, are added in increments during the fermentation. Theamount and the rate of addition of the nutrient may be determined byroutine experimentation. For example, the concentration of criticalnutrients in the fermentation medium may be monitored during thefermentation. Alternatively, more easily measured factors such as pH,dissolved oxygen, and the partial pressure of waste gases, such ascarbon dioxide, may be monitored. From these measured parameters, therate of nutrient addition may be determined. The amount of organicextractant used and its methods of addition in this mode is the same asthat used in the batchwise mode, described above. The amount of addedelectrolyte may be the same as in other fermentation modes.

Extraction of the product may be done downstream of the fermentor,rather than in situ. In this external mode, the extraction of thebutanol product into the organic extractant is carried out on thefermentation medium removed from the fermentor. The electrolyte may beadded to the fermentation medium removed from the fermentor. The amountof extractant used is about 20% to about 60% of the fermentor workingvolume; or 30% to about 60% of the fermentor working volume. Thefermentation medium may be removed from the fermentor continuously orperiodically, and the extraction of the butanol product by the organicextractant may be done with or without the removal of the cells from thefermentation medium. The cells may be removed from the fermentationmedium by means known in the art including, but not limited to,filtration or centrifugation. The electrolyte may be added to thefermentation medium before or after removal of the cells. Afterseparation of the fermentation medium from the extractant by meansdescribed above, the fermentation medium may be recycled into thefermentor, discarded, or treated for the removal of any remainingbutanol product. Similarly, the isolated cells may also be recycled intothe fermentor. After treatment to recover the butanol product, theextractant may be recycled for use in the extraction process.Alternatively, fresh extractant may be used. In this mode the extractantis not present in the fermentor, so the toxicity of the extractant ismuch less of a problem. If the cells are separated from the fermentationmedium before contacting with the extractant, the problem of extractanttoxicity may be further reduced. Furthermore, using this external modethere is less chance of forming an emulsion and evaporation of theextractant is minimized, alleviating environmental concerns.

Methods for Production of Butanol Using Extractive Fermentation withAdded Electrolyte

An improved method for the production of butanol is provided, wherein amicroorganism that has been genetically modified to produce butanol viaa biosynthetic pathway from at least one fermentable carbon source isgrown in a biphasic fermentation medium comprising an aqueous phase andi) a first water-immiscible organic extractant and optionally ii) asecond water-immiscible organic extractant, and the biphasicfermentation medium further comprises at least one electrolyte at aconcentration at least sufficient to increase the butanol partitioncoefficient relative to that in the presence of the salt concentrationof the basal fermentation medium. Such genetically modifiedmicroorganisms can be selected from bacteria, cyanobacteria, filamentousfungi and yeasts and include Escherichia coli, Lactobacillus plantarum,and Saccharomyces cerevisiae, for example The first water-immiscibleorganic extractant may be selected from the group consisting of C₁₂ toC₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fattyacids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides, and mixturesthereof, and the optional second water-immiscible organic extractant maybe selected from the group consisting of C₇ to C₂₂ alcohols, C₇ to C₂₂carboxylic acids, esters of C₇ to C₂₂ carboxylic acids, C₇ to C₂₂aldehydes, C₇ to C₂₂ fatty amides, and mixtures thereof, wherein thebiphasic fermentation medium comprises from about 10% to about 90% byvolume of the organic extractant. Alternatively, the biphasicfermentation medium may comprise from about 3% to about 60% by volume ofthe organic extractant, or from about 15% to about 50%. Themicroorganism is grown in the biphasic fermentation medium for a timesufficient to extract butanol into the extractant to form abutanol-containing organic phase. The at least sufficient concentrationof the electrolyte in the fermentation medium may be achieved by addingelectrolyte to the aqueous phase during the growth phase of themicroorganism, to the aqueous phase during the butanol production phase,to the aqueous phase when the butanol concentration in the aqueous phaseis inhibitory, to the first extractant, to the second extractant, or tocombinations thereof.

In one embodiment, the fermentation medium further comprises ethanol,and the butanol-containing organic phase can contain ethanol. Thebutanol-containing organic phase is then separated from the aqueousphase, as described above. Subsequently, the butanol is recovered fromthe butanol-containing organic phase, as described above.

Also provided is an improved method for the production of butanolwherein a microorganism that has been genetically modified to producebutanol via a biosynthetic pathway from at least one carbon source isgrown in a fermentation medium wherein the microorganism produces thebutanol into the fermentation medium to produce a butanol-containingfermentation medium. Such genetically modified microorganisms can beselected from bacteria, cyanobacteria, filamentous fungi and yeasts andinclude Escherichia coli, Lactobacillus plantarum, and Saccharomycescerevisiae, for example. At least one electrolyte is added to thefermentation medium to provide the electrolyte at a concentration atleast sufficient to increase the butanol partition coefficient relativeto that in the presence of the salt concentration of the basalfermentation medium. In one embodiment, the electrolyte may be added tothe fermentation medium when the microorganism growth phase slows. Inone embodiment, the electrolyte may be added to the fermentation mediumwhen the butanol production phase is complete. At least a portion of thebutanol-containing fermentation medium is contacted with a firstwater-immiscible organic extractant selected from the group consistingof C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ toC₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides,and mixtures thereof, and optionally ii) a second water-immiscibleorganic extractant selected from the group consisting of C₇ to C₂₂alcohols, C₇ to C₂₂carboxylic acids, esters of C₇ to C₂₂ carboxylicacids, C₇ to C₂₂ aldehydes, C₇ to C₂₂ fatty amides and mixtures thereof,to form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase. The butanol-containing organic phaseis then separated from the aqueous phase, as described above.Subsequently, the butanol is recovered from the butanol-containingorganic phase, as described above. At least a portion of the aqueousphase is returned to the fermentation medium. In one embodiment, thefermentation medium further comprises ethanol, and thebutanol-containing organic phase can contain ethanol.

Isobutanol may be produced by extractive fermentation with the use of amodified Escherichia coli strain in combination with an oleyl alcohol asthe organic extractant, as disclosed in U.S. patent application Ser. No.12/478,389. The method yields a higher effective titer for isobutanol(i.e., 37 g/L) compared to using conventional fermentation techniques(see Example 6 of U.S. patent application Ser. No. 12/478,389). Forexample, Atsumi et al. (Nature 451(3):86-90, 2008) report isobutanoltiters up to 22 g/L using fermentation with an Escherichia coli that wasgenetically modified to contain an isobutanol biosynthetic pathway. Thehigher butanol titer obtained with the extractive fermentation methoddisclosed in U.S. patent application Ser. No. 12/478,389 results atleast in part from the removal of the toxic butanol product from thefermentation medium, thereby keeping the level below that which is toxicto the microorganism. It is reasonable to assume that the presentextractive fermentation method employing the use of at least oneelectrolyte at a concentration in the fermentation medium at leastsufficient to increase the butanol partition coefficient relative tothat in the presence of the salt concentration of the basal fermentationmedium as defined herein would be used in a similar way and providesimilar results.

Butanol produced by the methods disclosed herein may have an effectivetiter of greater than 22 g per liter of the fermentation medium.Alternatively, the butanol produced by methods disclosed may have aneffective titer of at least 25 g per liter of the fermentation medium.Alternatively, the butanol produced by methods described herein may havean effective titer of at least 30 g per liter of the fermentationmedium. Alternatively, the butanol produced by methods described hereinmay have an effective titer of at least 37 g per liter of thefermentation medium.

The present methods are generally described below with reference to FIG.1 through FIG. 7.

Referring now to FIG. 1, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a fermentor 20, which contains at least one geneticallymodified microorganism (not shown) that produces butanol from afermentation medium comprising at least one fermentable carbon source.Optionally, electrolyte may be added as a separate stream (not shown) tothe fermentor. A stream of the first extractant 12 and a stream of theoptional second extractant 14 are introduced to a vessel 16, in whichthe first and second extractants are combined to form the combinedextractant 18. Optionally, electrolyte may be added (not shown) tostream 18, to vessel 16, to the stream of the first extractant 12, tothe stream of the second extractant 14, or to a combination thereof. Astream of the extractant 18 is introduced into the fermentor 20, inwhich contacting of the fermentation medium with the extractant to forma two-phase mixture comprising an aqueous phase and a butanol-containingorganic phase occurs. A stream 26 comprising both the aqueous andorganic phases is introduced into a vessel 38, in which separation ofthe aqueous and organic phases is performed to produce abutanol-containing organic phase 40 and an aqueous phase 42. Optionally,at least a portion of the aqueous phase 42 containing electrolyte isreturned (not shown) to fermentor 20 or another fermentor (not shown).The point(s) of addition of the electrolyte to the process are selectedsuch that the concentration of electrolyte in the aqueous phase 42 is atleast sufficient to increase the butanol partition coefficient relativeto that in the presence of the salt concentration of the basalfermentation medium.

Referring now to FIG. 2, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a fermentor 20, which contains at least one geneticallymodified microorganism (not shown) that produces butanol from afermentation medium comprising at least one fermentable carbon source.Optionally, electrolyte may be added as a separate stream (not shown) tothe fermentor. A stream of the first extractant 12 and a stream of theoptional second extractant 14 are introduced separately to the fermentor20, in which contacting of the fermentation medium with the extractantto form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase occurs. Optionally, electrolyte may beadded (not shown) to stream 12, to stream 14, or to a combinationthereof. A stream 26 comprising both the aqueous and organic phases isintroduced into a vessel 38, in which separation of the aqueous andorganic phases is performed to produce a butanol-containing organicphase 40 and an aqueous phase 42. Optionally, at least a portion of theaqueous phase 42 containing electrolyte is returned (not shown) tofermentor 20 or another fermentor (not shown). The point(s) of additionof the electrolyte to the process are selected such that theconcentration of electrolyte in the aqueous phase 42 is at leastsufficient to increase the butanol partition coefficient relative tothat in the presence of the salt concentration of the basal fermentationmedium.

Referring now to FIG. 3, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol usingin situ extractive fermentation. An aqueous stream 10 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a first fermentor 20, which contains at least onegenetically modified microorganism (not shown) that produces butanolfrom a fermentation medium comprising at least one fermentable carbonsource. Optionally, electrolyte may be added as a separate stream (notshown) to the fermentor. A stream of the first extractant 12 isintroduced to the fermentor 20, and a stream 22 comprising a mixture ofthe first extractant and the contents of fermentor 20 is introduced intoa second fermentor 24. A stream of the optional second extractant 14 isintroduced into the second fermentor 24, in which contacting of thefermentation medium with the extractant to form a two-phase mixturecomprising an aqueous phase and a butanol-containing organic phaseoccurs. Optionally, electrolyte may be added (not shown) to stream 12,to stream 22, to stream 14, to vessel 24, or to a combination thereof. Astream 26 comprising both the aqueous and organic phases is introducedinto a vessel 38, in which separation of the aqueous and organic phasesis performed to produce a butanol-containing organic phase 40 and anaqueous phase 42. Optionally, at least a portion of the aqueous phase 42containing electrolyte is returned (not shown) to fermentor 20 oranother fermentor (not shown). The point(s) of addition of theelectrolyte to the process are selected such that the concentration ofelectrolyte in the aqueous phase 42 is at least sufficient to increasethe butanol partition coefficient relative to that in the presence ofthe salt concentration of the basal fermentation medium.

Referring now to FIG. 4, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a fermentor 120, which contains at least one geneticallymodified microorganism (not shown) that produces butanol from afermentation medium comprising at least one fermentable carbon source.Optionally, electrolyte may be added as a separate stream (not shown) tothe fermentor. A stream of the first extractant 112 and a stream of theoptional second extractant 114 are introduced to a vessel 116, in whichthe first and second extractants are combined to form the combinedextractant 118. At least a portion, shown as stream 122, of thefermentation medium in fermentor 120 is introduced into vessel 124.Optionally, electrolyte may be added (not shown) to stream 112, tostream 114, to vessel 116, to stream 118, to vessel 124, or to acombination thereof. A stream of the extractant 118 is also introducedinto vessel 124, in which contacting of the fermentation medium with theextractant to form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase occurs. A stream 126 comprising boththe aqueous and organic phases is introduced into a vessel 138, in whichseparation of the aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142. At leasta portion of the aqueous phase 142 containing electrolyte is returned tofermentor 120, or optionally to another fermentor (not shown). Thepoint(s) of addition of the electrolyte to the process are selected suchthat the concentration of electrolyte in the aqueous phase 142 is atleast sufficient to increase the butanol partition coefficient relativeto that in the presence of the salt concentration of the basalfermentation medium.

Referring now to FIG. 5, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a fermentor 120, which contains at least one geneticallymodified microorganism (not shown) that produces butanol from afermentation medium comprising at least one fermentable carbon source.Optionally, electrolyte may be added as a separate stream (not shown) tothe fermentor. A stream of the first extractant 112 and a stream of thesecond extractant 114 are introduced separately to a vessel 124, inwhich the first and second extractants are combined to form the combinedextractant. Optionally, electrolyte may be added (not shown) to stream112, to stream 114, to stream 122, to vessel 124, or to combinationsthereof. At least a portion, shown as stream 122, of the fermentationmedium in fermentor 120 is also introduced into vessel 124, in whichcontacting of the fermentation medium with the extractant to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase occurs. A stream 126 comprising both the aqueous andorganic phases is introduced into a vessel 138, in which separation ofthe aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142. At leasta portion of the aqueous phase 142 containing electrolyte is returned tofermentor 120, or optionally to another fermentor (not shown). Thepoint(s) of addition of the electrolyte to the process are selected suchthat the concentration of electrolyte in the aqueous phase 142 is atleast sufficient to increase the butanol partition coefficient relativeto that in the presence of the salt concentration of the basalfermentation medium.

Referring now to FIG. 6, there is shown a schematic representation ofone embodiment of processes for producing and recovering butanol inwhich extraction of the product is performed downstream of thefermentor, rather than in situ. An aqueous stream 110 of at least onefermentable carbon source, optionally containing electrolyte, isintroduced into a fermentor 120, which contains at least one geneticallymodified microorganism (not shown) that produces butanol from afermentation medium comprising at least one fermentable carbon source.Optionally, electrolyte may be added as a separate stream (not shown) tothe fermentor. A stream of the first extractant 112 is introduced to avessel 128, and at least a portion, shown as stream 122, of thefermentation medium in fermentor 120 is also introduced into vessel 128.Optionally, electrolyte may be added (not shown) to stream 122, tostream 112, to vessel 128, or to a combination thereof. A stream 130comprising a mixture of the first extractant and the contents offermentor 120 is introduced into a second vessel 132. Optionally,electrolyte may be added (not shown) to stream 130, to stream 114, tovessel 132, or to a combination thereof. A stream of the optional secondextractant 114 is introduced into the second vessel 132, in whichcontacting of the fermentation medium with the extractant to form atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase occurs. A stream 134 comprising both the aqueous andorganic phases is introduced into a vessel 138, in which separation ofthe aqueous and organic phases is performed to produce abutanol-containing organic phase 140 and an aqueous phase 142. At leasta portion of the aqueous phase 142 containing electrolyte is returned tofermentor 120, or optionally to another fermentor (not shown). Thepoint(s) of addition of the electrolyte to the process are selected suchthat the concentration of electrolyte in the aqueous phase 142 is atleast sufficient to increase the butanol partition coefficient relativeto that in the presence of the salt concentration of the basalfermentation medium.

The extractive processes described herein can be run as batch processesor can be run in a continuous mode where fresh extractant is added andused extractant is pumped out such that the amount of extractant in thefermentor remains constant during the entire fermentation process. Suchcontinuous extraction of products and byproducts from the fermentationcan increase effective rate, titer and yield.

In yet another embodiment, it is also possible to operate theliquid-liquid extraction in a flexible co-current or, alternatively,counter-current way that accounts for the difference in batch operatingprofiles when a series of batch fermentors are used. In this scenariothe fermentors are filled with fermentable mash which provides at leastone fermentable carbon source and microorganism in a continuous fashionone after another for as long as the plant is operating. Referring toFIG. 7, once Fermentor F100 fills with mash and microorganism, the mashand microorganism feeds advance to Fermentor F101 and then to FermentorF102 and then back to Fermentor F100 in a continuous loop. Electrolytemay be added (not shown) to one or more Fermentors, to the streamentering the Fermentor, to the stream exiting the fermentor, or acombination thereof. The fermentation in any one fermentor begins oncemash and microorganism are present together and continues until thefermentation is complete. The mash and microorganism fill time equalsthe number of fermentors divided by the total cycle time (fill, ferment,empty and clean). If the total cycle time is 60 hours and there are 3fermentors then the fill time is 20 hours. If the total cycle time is 60hours and there are 4 fermentors then the fill time is 15 hours.

Adaptive co-current extraction follows the fermentation profile assumingthe fermentor operating at the higher broth phase titer can utilize theextracting solvent stream richest in butanol concentration and thefermentor operating at the lowest broth phase titer will benefit fromthe extracting solvent stream leanest in butanol concentration. Forexample, referring again to FIG. 7, consider the case where FermentorF100 is at the start of a fermentation and operating at relatively lowbutanol broth phase (B) titer, Fermentor F101 is in the middle of afermentation operating at relatively moderate butanol broth phase titerand Fermentor F102 is near the end of a fermentation operating atrelatively high butanol broth phase titer. In this case, lean extractingsolvent (S), with minimal or no extracted butanol, can be fed toFermentor F100, the “solvent out” stream (S′) from Fermentor F100 havingan extracted butanol component can then be fed to Fermentor F101 as its“solvent in” stream and the solvent out stream from F101 can then be fedto Fermentor F102 as its solvent in stream. The solvent out stream fromF102 can then be sent to be processed to recover the butanol present inthe stream. The processed solvent stream from which most of the butanolis removed can be returned to the system as lean extracting solvent andwould be the solvent in feed to Fermentor F100 above.

As the fermentations proceed in an orderly fashion the valves in theextracting solvent manifold can be repositioned to feed the leanestextracting solvent to the fermentor operating at the lowest butanolbroth phase titer. For example, assume (a) Fermentor F102 completes itsfermentation and has been reloaded and fermentation begins anew, (b)Fermentor F100 is in the middle of its fermentation operating atmoderate butanol broth phase titer and (c) Fermentor F101 is near theend of its fermentation operating at relatively higher butanol brothphase titer. In this scenario the leanest extracting solvent would feedF102, the extracting solvent leaving F102 would feed Fermentor F100 andthe extracting solvent leaving Fermentor F100 would feed Fermentor F101.

The advantage of operating this way can be to maintain the broth phasebutanol titer as low as possible for as long as possible to realizeimprovements in productivity. Additionally, it can be possible to dropthe temperature in the other fermentors that have progressed furtherinto fermentation that are operating at higher butanol broth phasetiters. The drop in temperature can allow for improved tolerance to thehigher butanol broth phase titers.

Advantages of the Present Methods

The present extractive fermentation methods provide butanol known tohave an energy content similar to that of gasoline and which can beblended with any fossil fuel. Butanol is favored as a fuel or fueladditive as it yields only CO₂ and little or no SO_(X) or NO_(X) whenburned in the standard internal combustion engine. Additionally, butanolis less corrosive than ethanol, the most preferred fuel additive todate.

In addition to its utility as a biofuel or fuel additive, the butanolproduced according to the present methods has the potential of impactinghydrogen distribution problems in the emerging fuel cell industry. Fuelcells today are plagued by safety concerns associated with hydrogentransport and distribution. Butanol can be easily reformed for itshydrogen content and can be distributed through existing gas stations inthe purity required for either fuel cells or vehicles. Furthermore, thepresent methods produce butanol from plant derived carbon sources,avoiding the negative environmental impact associated with standardpetrochemical processes for butanol production.

Advantages of the present methods include the feasibility of producingbutanol at net effective rate, titer, and yield that are significantlyhigher and more economical than the threshold levels of butanol obtainedby a two phase extractive fermentation process without the addition ofat least one electrolyte at a concentration at least sufficient toincrease the butanol partition coefficient relative to that in thepresence of the salt concentration of the basal fermentation medium. Thepresent method can also reduce the net amount of fresh or recycledextractant needed to achieve a desired level of butanol production froma batch fermentation.

EXAMPLES

The present invention is further defined in the following examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

Materials

The following materials were used in the examples. All commercialreagents were used as received.

All solvents were obtained from Sigma-Aldrich (St. Louis, Mo.) and wereused without further purification. The oleyl alcohol used was technicalgrade, which contained a mixture of oleyl alcohol (65%) and higher andlower fatty alcohols. Isobutanol (purity 99.5%) was obtained fromSigma-Aldrich and was used without further purification. Sodium sulfate(Na₂SO₄, CAS 7757-82-6, greater than 99% purity) was obtained fromSigma-Aldrich (St. Louis, Mo.). Sodium chloride (NaCl, CAS 7647-14-5,Technical grade) was purchased from EMD Chemicals, Inc. (Gibbstown,N.J.).

General Methods

Optical density reading for measuring microorganism cell concentrationwas done using a Thermo Electron Corporation Helios Alphaspectrophotometer. Measurements were typically done using a wavelengthof 600 nanometers.

Glucose concentration in the culture broth was measured rapidly using a2700 Select Biochemistry Analyzer (YSI Life Sciences, Yellow Springs,Ohio). Culture broth samples were centrifuged at room temperature for 2minutes at 13,200 rpm in 1.8 mL Eppendorf tubes, and the aqueoussupernatant analyzed for glucose concentration. The analyzer performed aself-calibration with a known glucose standard before assaying each setof fermentor samples; an external standard was also assayed periodicallyto ensure the integrity of the culture broth assays. The analyzerspecifications for the analysis were as follows:

Sample size: 15 μL

Black probe chemistry: dextrose

White probe chemistry: dextrose

Isobutanol and glucose concentrations in the aqueous phase were measuredby HPLC (Waters Alliance Model, Milford, Mass. or Agilent 1200 Series,Santa Clara, Calif.) using a BioRad Aminex HPX-87H column, 7.8 mm×300mm, (Bio-Rad laboratories, Hercules, Calif.) with appropriate guardcolumns, using 0.01 N aqueous sulfuric acid, isocratic, as the eluant.The sample was passed through a 0.2 μm centrifuge filter (Nanosep MFmodified nylon) into an HPLC vial. The HPLC run conditions were asfollows:

Injection volume: 10 μL

Flow rate: 0.60 mL/minute

Run time: 40 minutes

Column Temperature: 40° C.

Detector: refractive index

Detector temperature: 35° C.

UV detection: 210 nm, 8 nm bandwidth

After the run, concentrations in the sample were determined fromstandard curves for each of the compounds. The retention times were 32.6and 9.1 minutes for isobutanol and glucose, respectively.

Isobutanol and ethanol in the organic extractant phase was measuredusing Gas Chromatography (GC) as described below.

The following GC method was used to determine the amount of isobutanoland ethanol in the organic phase. The GC method utilized a J&WScientific DB-WAXETR column (50 m×0.32 mm ID, 1 μm film) from AgilentTechnologies (Santa Clara, Calif.). The carrier gas was helium at a flowrate of 4 mL/min with constant head pressure; injector split was 1:5 at250° C.; oven temperature was 40° C. for 5 min, 40° C. to 230° C. at 10°C./min, and 230° C. for 5 min. Flame ionization detection was used at250° C. with 40 mL/min helium makeup gas. Culture broth samples werecentrifuged before injection. The injection volume was 1.0 μL.Calibrated standard curves were generated for ethanol and isobutanol.Under these conditions, the isobutanol retention time was 9.9 minutes,and the retention time for ethanol was 8.7 minutes.

Construction of an E. coli Strain Having Deletions of pflB, frdB, IdhA,and adhE Genes

Provided herein is a suitable method for deleting pflB, frdB, IdhA, andadhE genes from E. coli. The Keio collection of E. coli strains (Baba etal., Mol. Syst. Biol., 2:1-11, 2006) was used for production of eight ofthe knockouts. The Keio collection (available from NBRP at the NationalInstitute of Genetics, Japan) is a library of single gene knockoutscreated in strain E. coli BW25113 by the method of Datsenko and Wanner(Datsenko, K. A. & Wanner, B. L., Proc Natl Acad. Sci., USA, 97:6640-6645, 2000). In the collection, each deleted gene was replaced witha FRT-flanked kanamycin marker that was removable by Flp recombinase.The E. coli strain carrying multiple knockouts was constructed by movingthe knockout-kanamycin marker from the Keio donor strain bybacteriophage P1 transduction to a recipient strain. After each P1transduction to produce a knockout, the kanamycin marker was removed byFlp recombinase. This markerless strain acted as the new recipientstrain for the next P1 transduction. One of the described knockouts wasconstructed directly in the strain using the method of Datsenko andWanner (supra) rather than by P1 transduction.

The 4KO E. coli strain was constructed in the Keio strain JW0886 byP1_(vir) transductions with P1 phage lysates prepared from three Keiostrains. The Keio strains used are listed below:

JW0886: the kan marker is inserted in the pflB

JW4114: the kan marker is inserted in the frdB

JW1375: the kan marker is inserted in the IdhA

JW1228: the kan marker is inserted in the adhE

[Sequences corresponding to the inactivated genes are: pflB (SEQ ID NO:71), frdB (SEQ ID NO: 73), IdhA (SEQ ID NO: 77), adhE (SEQ ID NO: 75).]

Removal of the FRT-flanked kanamycin marker from the chromosome wasperformed by transforming the kanamycin-resistant strain with pCP20 anampicillin-resistant plasmid (Cherepanov and Wackernagel, supra)).Transformants were spread onto LB plates containing 100 μg/mLampicillin. Plasmid pCP20 carries the yeast FLP recombinase under thecontrol of the λ_(PR) promoter and expression from this promoter iscontrolled by the cI857 temperature-sensitive repressor residing on theplasmid. The origin of replication of pCP20 is alsotemperature-sensitive.

Removal of the loxP-flanked kanamycin marker from the chromosome wasperformed by transforming the kanamycin-resistant strain with pJW168 anampicillin-resistant plasmid (Wild et al., Gene. 223:55-66, 1998)harboring the bacteriophage P1 Cre recombinase. Cre recombinase (Hoess,R. H. & Abremski, K., supra) meditates excision of the kanamycinresistance gene via recombination at the loxP sites. The origin ofreplication of pJW168 is the temperature-sensitive pSC101. Transformantswere spread onto LB plates containing 100 μg/mL ampicillin.

Strain JW0886 (ΔpflB::kan) was transformed with plasmid pCP20 and spreadon the LB plates containing 100 μg/mL ampicillin at 30° C. Ampicillinresistant transformants were then selected, streaked on the LB platesand grown at 42° C. Isolated colonies were patched onto the ampicillinand kanamycin selective medium plates and LB plates. Kanamycin-sensitiveand ampicillin-sensitive colonies were screened by colony PCR withprimers pflB CkUp (SEQ ID NO: 78) and pflB CkDn (SEQ ID NO: 79). A 10 μLaliquot of the PCR reaction mix was analyzed by gel electrophoresis. Theexpected approximate 0.4 kb PCR product was observed confirming removalof the marker and creating the “JW0886 markerless” strain. This strainhas a deletion of the pflB gene.

The “JW0886 markerless” strain was transduced with a P1_(vir) lysatefrom JW4114 (frdB::kan) and streaked onto the LB plates containing 25μg/mL kanamycin. The kanamycin-resistant transductants were screened bycolony PCR with primers frdB CkUp (SEQ ID NO: 80) and frdB CkDn (SEQ IDNO: 81). Colonies that produced the expected approximate 1.6 kb PCRproduct were made electrocompetent and transformed with pCP20 for markerremoval as described above. Transformants were first spread onto the LBplates containing 100 μg/mL ampicillin at 30° C. and ampicillinresistant transformants were then selected and streaked on LB plates andgrown at 42° C. Isolated colonies were patched onto ampicillin and thekanamycin selective medium plates and LB plates. Kanamycin-sensitive,ampicillin-sensitive colonies were screened by PCR with primers frdBCkUp (SEQ ID NO: 80) and frdB CkDn (SEQ ID NO: 81). The expectedapproximate 0.4 kb PCR product was observed confirming marker removaland creating the double knockout strain, “ΔpflB frdB”.

The double knockout strain was transduced with a P1_(vir) lysate fromJW1375 (ΔldhA::kan) and spread onto the LB plates containing 25 μg/mLkanamycin. The kanamycin-resistant transductants were screened by colonyPCR with primers IdhA CkUp (SEQ ID NO: 82) and IdhA CkDn (SEQ ID NO:83). Clones producing the expected 1.5 kb PCR product were madeelectrocompetent and transformed with pCP20 for marker removal asdescribed above. Transformants were spread onto LB plates containing 100μg/mL ampicillin at 30° C. and ampicillin resistant transformants werestreaked on LB plates and grown at 42° C. Isolated colonies were patchedonto ampicillin and kanamycin selective medium plates and LB plates.Kanamycin-sensitive, ampicillin-sensitive colonies were screened by PCRwith primers IdhA CkUp (SEQ ID NO: 82) and IdhA CkDn (SEQ ID NO: 83) fora 0.3 kb product. Clones that produced the expected approximate 0.3 kbPCR product confirmed marker removal and created the triple knockoutstrain designated “3KO” (ΔpflB frdB IdhA).

Strain “3 KO” was transduced with a P1_(vir) lysate from JW1228(ΔadhE::kan) and spread onto the LB plates containing 25 μg/mLkanamycin. The kanamycin-resistant transductants were screened by colonyPCR with primers adhE CkUp (SEQ ID NO: 84) and adhE CkDn (SEQ ID NO:85). Clones that produced the expected 1.6 kb PCR product were named 3KOadhE::kan. Strain 3KO adhE::kan was made electrocompetent andtransformed with pCP20 for marker removal. Transformants were spreadonto the LB plates containing 100 μg/mL ampicillin at 30° C. Ampicillinresistant transformants were streaked on the LB plates and grown at 42°C. Isolated colonies were patched onto ampicillin and kanamycinselective plates and LB plates. Kanamycin-sensitive,ampicillin-sensitive colonies were screened by PCR with the primers adhECkUp (SEQ ID NO: 84) and adhE CkDn (SEQ ID NO: 85). Clones that producedthe expected approximate 0.4 kb PCR product were named “4KO” (ΔpflB frdBIdhA adhE).

Construction of an E. Coli Production Host (Strain NGCI-031) Containingan Isobutanol Biosynthetic Pathway and Deletions of pflB, frdB, IdhA,and adhE Genes

A DNA fragment encoding sadB, a butanol dehydrogenase, (DNA SEQ ID NO:9;protein SEQ ID NO: 10) from Achromobacter xylosoxidans was amplifiedfrom A. xylosoxidans genomic DNA using standard conditions. The DNA wasprepared using a Gentra Puregene kit (Gentra Systems, Inc., Minneapolis,Minn.; catalog number D-5500A) following the recommended protocol forgram negative organisms. PCR amplification was done using forward andreverse primers N473 and N469 (SEQ ID NOs: 86 and 87), respectively withPhusion High Fidelity DNA Polymerase (New England Biolabs, Beverly,Mass.). The PCR product was TOPO-Blunt cloned into pCR4 BLUNT(Invitrogen) to produce pCR4Blunt::sadB, which was transformed into E.coli Mach-1 cells. Plasmid was subsequently isolated from four clones,and the sequence verified.

The sadB coding region was then cloned into the vector pTrc99a (Amann etal., Gene 69: 301-315, 1988). The pCR4Blunt::sadB was digested withEcoRI, releasing the sadB fragment, which was ligated withEcoRI-digested pTrc99a to generate pTrc99a::sadB. This plasmid wastransformed into E. coli Mach 1 cells and the resulting transformant wasnamed Mach1/pTrc99a::sadB. The activity of the enzyme expressed from thesadB gene in these cells was determined to be 3.5 mmol/min/mg protein incell-free extracts when analyzed using isobutyraldehyde as the standard.

The sadB gene was then subcloned into pTrc99A::budB-ilvC-ilvD-kivD asdescribed below. The pTrc99A::budB-ilvC-ilvD-kivD is the pTrc-99aexpression vector carrying an operon for isobutanol expression(described in Examples 9-14 the of U.S. Patent Application PublicationNo. 20070092957). The first gene in the pTrc99A::budB-ilvC-ilvD-kivDisobutanol operon is budB encoding acetolactate synthase from Klebsiellapneumoniae ATCC 25955, followed by the ilvC gene encoding acetohydroxyacid reductoisomerase from E. coli. This is followed by ilvD encodingacetohydroxy acid dehydratase from E. coli and lastly the kivD geneencoding the branched-chain keto acid decarboxylase from L. lactis.

The sadB coding region was amplified from pTrc99a::sadB using primersN695A (SEQ ID NO: 88) and N696A (SEQ ID NO: 89) with Phusion HighFidelity DNA Polymerase (New England Biolabs, Beverly, Mass.).Amplification was carried out with an initial denaturation at 98 C. for1 min, followed by 30 cycles of denaturation at 98° C. for 10 sec,annealing at 62° C. for 30 sec, elongation at 72° C. for 20 sec and afinal elongation cycle at 72° C. for 5 min, followed by a 4° C. hold.Primer N695A contained an AvrII restriction site for cloning and a RBSupstream of the ATG start codon of the sadB coding region. The N696Aprimer included an XbaI site for cloning. The 1.1 kb PCR product wasdigested with AvrII and XbaI (New England Biolabs, Beverly, Mass.) andgel purified using a Qiaquick Gel Extraction Kit (Qiagen Inc., Valencia,Calif.)). The purified fragment was ligated withpTrc99A::budB-ilvC-ilvD-kivD, that had been cut with the samerestriction enzymes, using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The ligation mixture was incubated at 16° C. overnight and thentransformed into E. coli Mach 1™ competent cells (Invitrogen) accordingto the manufacturer's protocol. Transformants were obtained followinggrowth on the LB agar with 100 μg/ml ampicillin. Plasmid DNA from thetransformants was prepared with QIAprep Spin Miniprep Kit (Qiagen Inc.,Valencia, Calif.) according to manufacturer's protocols. The resultingplasmid was called pTrc99A::budB-ilvC-ilvD-kivD-sadB.

Electrocompetent cells of the 4KO strains were prepared as described andtransformed with pTrc99A::budB-ilvC-ilvD-kivD-sadB (“pBCDDB”).Transformants were streaked onto LB agar plates containing 100 μg/mLampicillin. The resulting strain carrying plasmidpTrc99A::budB-ilvC-ilvD-kivD-sadB with 4KO (designated strain NGCI-031)was used for fermentation studies in the indicated Examples.

Example 1 Effect of Electrolyte Concentration on the PartitionCoefficient (K_(p))

The purpose of this Example was to evaluate the effect of electrolyteconcentrations in the fermentation medium on the partition coefficient(K_(p)) of isobutanol when oleyl alcohol was used as the extractant. Thebasal fermentation medium (BFM) typically used in E. coli fermentationswas used as the fermentation medium in this Example. The BFM compositionis shown in Table 2.

TABLE 2 BFM Composition Concentration (g/L) Concentration Components oras indicated (millimoles/L; mM) Potassium phosphate 13.3 97.73 monobasicAmmonium phosphate 4.0 30.28 dibasic Citric acid monohydrate 1.7 8.09Magnesium sulfate 2.0 8.11 heptahydrate Trace Elements (mL/L) 10.0 —Thiamine Hydrochloride 4.5 — (mg/L) Yeast Extract 5.0 — Sigma Antifoam204 0.20 — (mL/L) Glucose 30.0 —

The trace elements solution used in the above medium was prepared asfollows. Ingredients listed below were added in the order listed and thesolution is heated to 50-60° C. until all the components are completelydissolved. Ferric citrate was added slowly after other ingredients werein solution. The solution was filter sterilized using 0.2 micronfilters.

EDTA 0.84 g/L (Ethylenediaminetetra acetic acid) Cobalt dichloridehexahydrate 0.25 g/L (cobalt chloride 6-hydrate) Manganese dichloridetetrahydrate  1.5 g/L (manganese chloride 4-hydrate) Cupric chloridedihydrate 0.15 g/L Boric acid (H₃BO₃) 0.30 g/L Sodium molybdatedihydrate 0.25 g/L Zinc acetate dihydrate 1.30 g/L Ferric citrate 10.0g/L

The initial level of total salts (sum of potassium phosphate monobasic,ammonium phosphate dibasic, citric acid monohydrate, and magnesiumsulfate heptahydrate) in BFM as shown in Table 2 is calculated to beabout 144.2 mM. Since an E. coli biocatalyst was used in the Examplesshown below, betaine hydrochloride (Sigma-Aldrich) at 0.31 g/L (2mmoles/L) was added to the basal fermentation medium since it isreported in the literature (Cosquer A, et al; 1999; Appl EnvironMicrobiol 65:3304-3311) to improve the salt tolerance of E. coli.

The following experimental procedure was used to generate the data inTables 3 and 4. In these K_(p) measurement experiments, a specifiedamount of electrolyte as sodium sulfate (Na₂SO₄) or sodium chloride(NaCl) was added to the basal fermentation medium. To 30 mL of theelectrolyte-supplemented BFM, 10 mL of isobutanol rich oleyl alcohol(OA) extractant containing 168 g/L of isobutanol was added and mixedvigorously for about 4-8 hours at 30° C. with shaking at 250 rpm in atable top shaker (Innova 4230, New Brunswick scientific, Edison, N.J.)to reach equilibrium between the two phases. The aqueous and organicphases in each flask were separated by decantation. The aqueous phasewas centrifuged (2 minutes on 13,000 rpm with an Eppendorf centrifugemodel 5415R) to remove residual extractant phase and the supernatantanalyzed for glucose and isobutanol by HPLC. Analysis of isobutanollevels in the aqueous phase after 4 hrs of shaking was similar to thatobtained following 8 hrs of mixing suggesting that equilibration betweenthe two phases was attained within 4 hours. The intent was to show thatfurther mixing beyond 4 hours did not change K_(p).

Partition coefficients (K_(p)) for the isobutanol distribution betweenthe organic and aqueous phases were calculated from the known amount ofisobutanol added to the flask and the isobutanol concentration datameasured for the aqueous phase. The concentration of isobutanol in theextractant phase was determined by the mass balance. The partitioncoefficient was determined as the ratio of the isobutanol concentrationsin the organic and the aqueous phases, i.e.,K_(p)=[Isobutanol]_(Organic phase)/[isobutanol]_(Aqueous phase). Eachdata point corresponding to a specified level of electrolyte as shown inTable 3 and Table 4 was repeated twice and values for K_(p) reported asthe average of the two flasks.

TABLE 3 Effect of Sodium Sulfate (Na₂SO₄) Concentration on K_(p) ofisobutanol Total initial Amount of Total amount of concentration ofsodium sulfate salts in salts in BFM added to BFM experiment (Table 2)Na₂SO₄ moles/L moles/L (a) (moles/L) (b) (a) + (b) K_(p) 0.14 0.00 0.144.80 0.14 0.03 0.17 5.03 0.14 0.07 0.21 5.25 0.14 0.15 0.29 5.78 0.140.22 0.36 6.37 0.14 0.29 0.43 7.12 0.14 0.44 0.58 8.34 0.14 0.67 0.8110.50 0.14 1.00 1.14 15.95 0.14 1.33 1.47 24.68 0.14 2.00 2.14 60.99

TABLE 4 Effect of Sodium Chloride (NaCl) Concentration on K_(p) ofisobutanol Total initial Amount of Total amount of concentration ofsodium chloride salt in salts in BFM added to BFM experiment (Table 2)NaCl moles/L moles/L (a) (moles/L) (b) (a) + (b) K_(p) 0.14 0.00 0.144.87 0.14 0.01 0.15 4.87 0.14 0.04 0.18 4.89 0.14 0.07 0.21 4.95 0.140.11 0.25 5.00 0.14 0.14 0.28 5.00 0.14 0.21 0.35 5.22 0.14 0.33 0.475.04 0.14 0.67 0.81 5.91 0.14 1.00 1.14 6.88 0.14 1.33 1.47 8.06

Results from Table 3 and 4 demonstrate that supplementation of theaqueous fermentation medium with the electrolytes Na₂SO₄ and NaClresulted in higher K_(p) for isobutanol in a two phase system with oleylalcohol as the extractant phase.

Example 2 Effect of Electrolyte Supplementation on Growth Rate of E.Coli

To evaluate the effect of electrolytes such as Na₂SO₄ on growthproperties of the biocatalyst, 4KO E. coli strain, was grown in shakeflasks in BFM medium supplemented with 0.31 g/l of betaine hydrochlorideand different levels of Na₂SO₄ (0-284 g/L) at 30° C., 250 RPM in Innovatable top shakers. From a frozen vial, 25 mL of seed culture was grownin Difco LB broth, Miller medium, purchased from BD Laboratories (Becton& Dickinson and Company, Sparks, Md., 21152, USA) at 30° C., 200 RPM. 1mL of this seed culture was added to shake flasks containing 30 mL ofBFM medium supplemented with 0.31 g/L of betaine hydrochloride andvarying levels of Na₂SO₄. Samples were withdrawn at defined time pointsto monitor biomass growth as measured by OD₆₀₀. Growth rates werecalculated from the biomass time profiles by fitting exponential growthrate equations.

TABLE 5 Effect of Na₂SO₄ on growth rate of 4KO E. coli strain Totalinitial Total amount concentration Concentration of of salts in of saltsin BFM Na₂SO₄ (mole/L) experiment (Table 2) added to BFM moles/L E. coliGrowth moles/L (a) (b) (a) + (b) Rate (μ) hr⁻¹ 0.14 0.00 0.14 0.79 0.140.03 0.17 0.79 0.14 0.07 0.21 0.79 0.14 0.15 0.29 0.79 0.14 0.22 0.360.74 0.14 0.29 0.43 0.69 0.14 0.44 0.58 0.60 0.14 0.67 0.81 0.55 0.141.00 1.14 0.14 0.14 1.33 1.47 Negligible growth 0.14 2.00 2.14Negligible Growth

The growth rate data shown in Table 5 suggest that the biocatalyst cantolerate salt levels as high as about 0.67 M Na₂SO₄ (total salt level of0.81 M) with a 30% loss of growth rate compared to no electrolytecontrol. However, there is a significant drop (greater than about 80%)in growth rate at 1M salt concentration. Data in Table 3 shows that at0.67 M concentration of Na₂SO₄, K_(p) for butanol increases by two-foldcompared to no salt addition control when oleyl-alcohol is present asthe extractant phase. Thus the net overall effect of electrolyteaddition to a 2-phase extractive fermentation using a recombinantbutanol producing microorganism can be unpredictable since electrolyteson one hand can inhibit cell growth (Table 3) but on the other canincrease the partitioning coefficient of toxic butanol product whichcould alleviate its toxic effects on the microorganism.

Example 3 Effect of Electrolyte Addition on Rate, Titer, and Yield ofButanol Production in a Two Phase Extractive Fermentation Process

The purpose of this example was to demonstrate the advantages of theaddition of at least a sufficient amount of electrolyte to the aqueousphase of a two-phase extractive fermentation in which butanol isproduced by a recombinant microorganism, a strain of Escherichia coli(NGCI-031) that contains an isobutanol biosynthetic pathway. Theextractive fermentation uses oleyl alcohol as the water-immiscible,organic extractant.

The Escherichia coli strain NGCI-031 was constructed as described in theGeneral Methods Section herein above. All seed cultures for inoculumpreparation were grown in Luria-Bertani (LB) medium with ampicillin (100mg/L) as the selection antibiotic. The fermentation medium used was asemi-synthetic medium supplemented with 2 mmoles/L of betainehydrochloride, the composition of which is given in Table 6.

TABLE 6 Fermentation Medium Composition Amount Ingredient Amount/L(mmoles/L) Phosphoric Acid 85% 0.75 mL 14.4 Sulfuric Acid (18M) 0.30 mL5.60 Balch's w/Cobalt - 1000X 1.00 mL NA (composition given in Table 7)Potassium Phosphate Monobasic 1.40 g 10.30 Citric Acid Monohydrate 2 g9.50 Magnesium Sulfate, heptahydrate 2 g 8.10 Ferric Ammonium Citrate0.33 g 1.25 Calcium chloride, dihydrate 0.20 g 1.36 Yeast Extract^(a)5.00 g Antifoam 204^(b) 0.20 mL Betaine Hydrochloride 0.32 gThiamince•HCl, 5 g/L stock 1.00 mL Ampicillin, 25 mg/mL stock 4.00 mLGlucose 50 wt % stock 33.3 mL ^(a)Obtained from BD Diagnostic Systems,Sparks, MD ^(b)Obtained from Sigma-Aldrich

TABLE 7 Balch's Modified Trace Metals - 1000X Ingredient Concentration(g/L) Citric Acid Monohydrate 40.0 MnSO₄•H₂O 30.0 NaCl 10.0 FeSO₄•7H₂O1.0 CoCl₂•6H₂O 1.0 ZnSO₄•7H₂O 1.5 CuSO₄•5H₂O 0.1 Boric Acid (H₃BO₃) 0.1Sodium Molybnate (NaMoO₄•2H₂O) 0.1

Ingredients 1-11 from Table 6 were added to water at the prescribedconcentration to make a final volume of 0.4 L in the fermentor. Thecontents of the fermentor were sterilized by autoclaving. Components12-14 were mixed, filter sterilized and added to the fermentor after theautoclaved medium had cooled. The total final volume of the fermentationmedium (the aqueous phase) was about 0.5 L following addition of 50 mlof seed inoculum.

Electrolyte in the form of Na₂SO₄ was added at 0 g/L, 40 g/L or 60 g/Lconcentrations to the medium before sterilization. Filter sterilizedsolutions of ampicillin, thiamine hydrochloride, and glucose were addedto the fermentor medium, post sterilization, to a final concentration of100 mg/L, 5 mg/L and 20 g/L respectively. Fermentations were run using a1 L autoclavable bioreactor, Bio Console ADI 1025 (Applikon, Inc,Holland) with a working volume of 900 mL. The temperature was maintainedat 30° C. during the entire fermentation and the pH was maintained at6.8 using ammonium hydroxide. Following inoculation of the sterilefermentation medium with seed culture (2-10 vol %), the fermentor wasoperated aerobically at a 30% dissolved oxygen (DO) set point with 0.3vvm of air flow by automatic control of the agitation rate (rpm). Oncethe desired optical density (OD₆₀₀) was reached (i.e., OD₆₀₀=10), theculture was induced with the addition of 0.4-0.5 mM isopropyl beta-D-1thiogalactopyranoside to overexpress the isobutanol biosyntheticpathway. Four hours post induction, fermentation conditions wereswitched to microaerobic conditions by decreasing the airflow to 0.13slpm and setting the DO set point to 3-5%. The shift to microaerobicconditions initiated isobutanol production while minimizing the amountof carbon going to biomass production, thereby uncoupling biomassformation from isobutanol production. Oleyl alcohol (about 250 mL) wasadded during the isobutanol production phase to alleviate the problem ofinhibition due to build up of isobutanol in the aqueous phase. Glucosewas added as a bolus (50 wt % stock solution) to the fermentor on a needbasis to keep levels of glucose between 20 g/L and 2 g/L.

Because efficient production of isobutanol requires microaerobicconditions to enable redox balance in the biosynthetic pathway, air wascontinuously supplied to the fermentor at 0.3 vvm. Continuous aerationled to significant stripping of isobutanol from the aqueous phase of thefermentor. To quantify the loss of isobutanol due to stripping, theoff-gas from the fermentor was directly sent to a mass spectrometer(Prima dB mass spectrometer, Thermo Electron Corp., Madison, Wis.) toquantify the amount of isobutanol in the gas stream. The isobutanolpeaks at mass to charge ratios of 74 or 42 were monitored continuouslyto quantify the amount of isobutanol in the gas stream.

For isobutanol production, the effective titer, the effective rate, andthe effective yield, all corrected for the isobutanol lost due tostripping, are shown below in tabular form (Table 8). Isobutanol in theaqueous phase was measured using the HPLC method described above herein.Isobutanol in the oleyl-alcohol extractant phase was measured using theGC method described above herein. Glucose levels were monitored usingHPLC and YSI as described above herein.

As can be seen from the results in Table 8, the use of electrolytes inan extractive fermentation for isobutanol production results insignificantly higher effective titer, effective rate, and effectiveyield compared to the case where no salt is added. The isobutanolproduct, which is toxic to the bacterial host, is continuously extractedinto the oleyl alcohol phase, decreasing its concentration in theaqueous phase, thereby reducing its toxicity to the microorganism.Additionally, unexpected improvement in the effective rate, effectivetiter, and effective yield is observed when salt is added to the medium.Addition of salts in principle could not only have a deleterious effecton the metabolism of the butanol producing biocatalyst but alsoalleviate the inhibitory effect of butanol by increasing Kp of butanolcompared to no salt addition control. The net effect of addition ofsalts in our 2-phase extractive system favors increased production andrecovery of butanol.

TABLE 8 Effect of Salts on Rate, Titer, and Yield of Butanol Productionin Example 3. Na₂SO₄ concentration Total amount of Kp added tofermentation salts in experiment Effective Effective Effective [Conc inOA medium in Table 6 (Table 6 + Na₂SO₄) Rate Titer Yield phase]/[Conc(moles/L) moles/L (g/L/hr) (g/L) (g/g) in Aq Phase] 0 0.05 0.09 6 0.063.1 0.28 0.33 0.14 9 0.10 4.5 0.42 0.47 0.14 9.4 0.12 5.4

Initial amount of salts in fermentation medium (Table 6) was about 0.05moles/L.

Example 4 Effect of Electrolyte Addition on Rate, Titer, and Yield ofButanol Production Coupled with Gas Stripping of Butanol DuringFermentation

In order to evaluate the effect of electrolyte addition on butanolproduction during aqueous phase fermentation without the addition ofoleyl alcohol extractant, Example 3 was repeated, except thatoleyl-alcohol was not added to any of the fermentors. In this Example,gas stripping of butanol from the aqueous phase was prevalent due to airsparging of the fermentors. The amount of butanol stripped to theoff-gas was quantified as in Example 3 by using a mass-spec. Effectiverate, titer, and yield, all corrected for butanol lost due to strippingare shown below in Table 9.

TABLE 9 Effect of salts on rate, titer, and yield of butanol productioncoupled with gas stripping of butanol during fermentation for Example 4Na₂SO₄ concentration Total amount of “grams” of added to fermentationsalts in experiment Effective Effective Effective isobutanol medium inTable 6 (Table 6 + Na₂SO₄) Rate Titer Yield lost due to (moles/L)moles/L (g/l/hr) (g/L) (g/g) stripping 0 0.05 0.08 6.0 0.11 2.55 0.280.33 0.16 10.6 0.17 4.25 0.42 0.47 0.17 10.9 0.14 5.01 [Initial amountof salts in fermentation medium (Table 9) was about 0.05 moles/L]

Results from Table 9 show that addition of electrolyte to the aqueousphase increases rate, titer and yield of butanol production in theabsence of oleyl alcohol by increasing the stripping rate of isobutanol.Grams of butanol stripped are almost two fold higher in the presence ofsalt compared to the case with no addition of electrolyte.

Although particular embodiments of the present invention have beendescribed in the foregoing description, it will be understood by thoseskilled in the art that the invention is capable of numerousmodifications, substitutions, and rearrangements without departing fromthe spirit or essential attributes of the invention. Reference should bemade to the appended claims, rather than to the foregoing specification,as indicating the scope of the invention.

What is claimed is:
 1. A method for the production of butanolcomprising: a) providing a genetically modified microorganism thatproduces butanol from at least one fermentable carbon source; b) growingthe microorganism in a biphasic fermentation medium comprising anaqueous phase and i) a first water-immiscible organic extractantselected from the group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ toC₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fattyaldehydes, C₁₂ to C₂₂ fatty amides and mixtures thereof, and optionallyii) a second water-immiscible organic extractant selected from the groupconsisting of C₇ to C₂₂ alcohols, C₇ to C₂₂carboxylic acids, esters ofC₇ to C₂₂ carboxylic acids, C₇ to C₂₂ aldehydes, C₇ to C₂₂ fatty amides,and mixtures thereof, wherein the biphasic fermentation medium furthercomprises at least one electrolyte at a concentration at leastsufficient to increase the butanol partition coefficient relative tothat in the presence of the salt concentration of the basal fermentationmedium, for a time sufficient to allow extraction of the butanol intothe organic extractant to form a butanol-containing organic phase; c)separating the butanol-containing organic phase from the aqueous phase;and d) optionally, recovering the butanol from the butanol-containingorganic phase to produce recovered butanol.
 2. The method of claim 1,wherein the electrolyte is added to the aqueous phase during the growthphase of the microorganism, to the aqueous phase during the butanolproduction phase, to the aqueous phase when the butanol concentration inthe aqueous phase is inhibitory, to the first extractant, to theoptional second extractant, or to combinations thereof.
 3. The method ofclaim 1, wherein the electrolyte is obtained from a fermentation medium.4. A method for the production of butanol comprising: a) providing agenetically modified microorganism that produces butanol from at leastone fermentable carbon source; b) growing the microorganism in afermentation medium wherein the microorganism produces the butanol intothe fermentation medium to produce a butanol-containing fermentationmedium; c) adding at least one electrolyte to the fermentation medium toprovide the electrolyte at a concentration at least sufficient toincrease the butanol partition coefficient relative to that in thepresence of the salt concentration of the basal fermentation medium; d)contacting at least a portion of the butanol-containing fermentationmedium with i) a first water-immiscible organic extractant selected fromthe group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fattyacids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂to C₂₂ fatty amides and mixtures thereof, and optionally ii) a secondwater-immiscible organic extractant selected from the group consistingof C₇ to C₂₂ alcohols, C₇ to C₂₂ carboxylic acids, esters of C₇ to C₂₂carboxylic acids, C₇ to C₂₂ aldehydes, C₇ to C₂₂ fatty amides, andmixtures thereof, to form a two-phase mixture comprising an aqueousphase and a butanol-containing organic phase; e) optionally, recoveringthe butanol from the butanol-containing organic phase; and f)optionally, returning at least a portion of the aqueous phase to thefermentation medium.
 5. The method of claim 4, wherein the electrolyteis added to the fermentation medium in step (c) when the microorganismgrowth phase slows.
 6. The method of claim 4, wherein the electrolyte isadded to the fermentation medium in step (c) when the butanol productionphase is complete.
 7. The method of claim 1, wherein said at least onefermentable carbon source is present in the fermentation medium andcomprises renewable carbon from agricultural feedstocks, algae,cellulose, hemicellulose, lignocellulose, or any combination thereof. 8.A composition comprising (a) a fermentation medium comprising butanol,water, at least one electrolyte at a concentration at least sufficientto increase the butanol partition coefficient ion medium, and agenetically modified microorganism that produces butanol from least onefermentable carbon source; b) a first water-immiscible organicextractant selected from the group consisting of C₁₂ to C₂₂ fattyalcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides and mixtures thereof;and c) optionally a second water-immiscible organic extractant selectedfrom the group consisting of C₇ to C₂₂ fatty alcohols, C₇ to C₂₂ fattyacids, esters of C₇ to C₂₂ fatty acids, C₇ to C₂₂ fatty aldehydes, C₇ toC₂₂ fatty amides and mixtures thereof; wherein said composition forms atwo-phase mixture comprising an aqueous phase and a butanol-containingorganic phase whereby butanol may be separated from the fermentationmedium of (a).
 9. The method of claim 1, wherein the electrolytecomprises a salt having a cation selected from the group consisting oflithium, sodium, potassium, rubidium, cesium, magnesium, calcium,strontium, barium, ammonium, phosphonium, and combinations thereof. 10.The method of claim 1, wherein the electrolyte comprises a salt havingan anion selected from the group consisting of sulfate, carbonate,acetate, citrate, lactate, phosphate, fluoride, chloride, bromide,iodide, and combinations thereof.
 11. The method of claim 1, wherein theelectrolyte is selected from the group consisting of sodium sulfate,sodium chloride, and combinations thereof.
 12. The method of claim 1,wherein the first extractant is selected from the group consisting ofoleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristylalcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid,stearic acid, methyl myristate, methyl oleate, lauric aldehyde,1-dodecanol, and mixtures thereof.
 13. The method of claim 1, whereinthe butanol is 1-butanol, 2-butanol, isobutanol, or mixtures thereof.14. The method of claim 1, wherein the fermentation medium furthercomprises ethanol, and the butanol-containing organic phase containsethanol.
 15. The method of claim 1, wherein the genetically modifiedmicroorganism comprises a modification which inactivates a competingpathway for carbon flow.
 16. The method of claim 1, wherein thegenetically modified microorganism does not produce acetone.
 17. Themethod of claim 1, wherein a portion of the butanol is concurrentlyremoved from the fermentation medium by a process comprising the stepsof: a) stripping butanol from the fermentation medium with a gas to forma butanol-containing gas phase; and b) recovering butanol from thebutanol-containing gas phase.
 18. The method of claim 1, wherein thegenetically modified microorganism is selected from the group consistingof bacteria, cyanobacteria, filamentous fungi, and yeast.
 19. The methodof claim 18, wherein the microorganism is selected from the groupconsisting of Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula, Kluyveromyces, Issatchenkia,and Saccharomyces.